WO2024081970A2 - Nouveaux oligonucléotides antisens contenant un morpholino hybride et un adn/arn (modifié) avec un lieur phosphorothioate (ps) pour le traitement du cancer et de troubles auto-immuns - Google Patents

Nouveaux oligonucléotides antisens contenant un morpholino hybride et un adn/arn (modifié) avec un lieur phosphorothioate (ps) pour le traitement du cancer et de troubles auto-immuns Download PDF

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WO2024081970A2
WO2024081970A2 PCT/US2023/077027 US2023077027W WO2024081970A2 WO 2024081970 A2 WO2024081970 A2 WO 2024081970A2 US 2023077027 W US2023077027 W US 2023077027W WO 2024081970 A2 WO2024081970 A2 WO 2024081970A2
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composition
stat3
aso
chimeric
seq
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PCT/US2023/077027
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English (en)
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Xuedong Liu
Marvin Caruthers
Kavitha SUDHEENDRAN
Gan Zhang
Xiaojuan Zhang
Yuefeng GAO
Gilson SANCHEZ
Cedric STAHEL
Ondrej KOSTOV
Balazs SCHAEFER
Mathias Bogetoft DANIELSEN
Marija CIBA
Saheli Ganguly
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The Regents Of The University Of Colorado, A Body Corporate
Vesicle Therapeutics Inc.
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Publication of WO2024081970A2 publication Critical patent/WO2024081970A2/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the instant application contains contents of the electronic sequence listing (90245-00871- Sequence-Listing.xml; Size: 70,848 bytes; and Date of Creation: October 16, 2023) is herein incorporated by reference in its entirety.
  • TECHNICAL FIELD The present invention is directed to methods and compositions for suppression of STAT3 mRNA and protein expression, as well as the modulation of STAT3 ⁇ and STAT3 ⁇ ⁇ isoforms via alternative splicing of STAT3 mRNA. Such methods and compositions are useful to treat, prevent, or inhibit the progression of disease conditions associated with STAT3, such as cancer, and in particular neoplastic growth, as well as autoimmune disorders.
  • STAT3 BACKGROUND Signal Transducer and Activator Of Transcription 3
  • STAT3 is a ubiquitously expressed transcription factor that is activated in response to growth factors and cytokines to promote cell growth and survival and modulate immune suppression.
  • STAT3 is activated by Tyrosine phosphorylation induced by tyrosine kinases (TKs) such as JAK2 or Src, which leads to dimerization of phosphorylated STAT3, nuclear translocation and binding to the STAT3 sites in the regulatory region of STAT3 target genes ( Figure 1).
  • TKs tyrosine kinases
  • STAT3 target genes are involved in cell proliferation (e.g., MYC, Cyclin D and VEGF) or anti-apoptosis (BCL-xl, BCL2, and MCL1) or epithelial to mesenchymal transition aka EMT (e.g., Twist and Vimentin).
  • EMT epithelial to mesenchymal transition
  • STAT3 activation is rapid and transient in response to environmental stimuli.
  • tumorigenesis aberrantly-active STAT3 is seen in a variety to tumors and has a causal link to tumor initiation, progression, metastasis and drug resistance. Beyond its roles in promoting tumor intrinsic properties, STAT3 also plays an important role in driving host immune suppression to allow tumor cells to evade immune surveillance.
  • STAT3 signaling has been linked to the promotion of a suppressive myeloid-derived suppressor cell (MDSC) phenotype and protumor macrophage polarization in part by regulating a cohort of genes that are involved in immunosuppression.
  • a small fraction of STAT3 that is prevalently phosphorylated at Ser727 has also been found to be imported into mitochondria.
  • Mitochondrial-targeted STAT3 has been postulated to play a role in maintaining glycolytic metabolism seen in cancer cells.
  • STAT3 is an oncogenic transcription factor that fuels tumor expansion through both tumor-intrinsic and host immune suppression mechanisms. It has been well established that STAT3 is a validated drug target for several types of cancers in both preclinical and clinical studies.
  • STAT3 Different therapeutic modalities have been employed to inhibit STAT3. So far no STAT3-specific therapeutics have been approved. Small molecule inhibitors targeting STAT3 have been in development for years but have failed to achieve much clinical success. Specifically, the systemic toxicity of targeting STAT3 may be attributed to the fact that STAT3 is essential for normal cellular functions.
  • the STAT3 gene encodes two main isoforms, STAT3 ⁇ and STAT3 ⁇ produced by alternative splicing. The structure difference of STAT3 ⁇ and STAT3 ⁇ is at the C-terminal region. Specifically, STAT3 ⁇ ⁇ is lacking 55 amino acids at the C-terminal of STAT3 ⁇ ⁇ and is replaced with a 7 amino-acid sequence.
  • STAT3 ⁇ is the predominant, constitutively active form in cancer, driving various oncogenic activities. Consistent with its essential role, loss of STAT3 ⁇ is lethal in mice. In contrast, STAT3 ⁇ , which lacks the C- terminal transactivation domain, has distinct regulatory and transcriptional functions. Isoform- specific mice knockout showed that STAT3 ⁇ is not required for viability and can avert embryonic lethality in total STAT3 knock-out mice. Recent research suggested that STAT3 ⁇ is an oncoprotein while STAT3 ⁇ is a tumor suppressor. It was hypothesized that STAT3 ⁇ protein dimerization is the key for pro-growth pathway, via STAT3 ⁇ ⁇ ⁇ heterodimer has the opposite effect.
  • Oligodeoxynucleotides that bind STAT3 and serve as a decoy for activated STAT3 have entered phase I clinical trial.
  • Antisense oligonucleotides have been used to modulate gene expression for decades. Recent advances in oligonucleotide chemistry, selectivity, and potency have led to enhanced stability, safety and pharmacokinetics properties resulting in the approval of several ASO drugs for chronic diseases.
  • Antisense oligonucleotides for degradation of STAT3 mRNA have been developed for the treatment of cancer and one ASO entered phase II clinical trials. However, these antisense oligonucleotides only use a DNA/RNA(modified) phosphorothioate structure motifs ( Figure 2).
  • Antisense oligonucleotides with morpholine motifs have been advanced to marketed drugs.
  • all morpholine antisense oligonucleotides in clinical development have morpholine phosphoramidate nucleotides (PMO), and their synthesis is not amenable to the synthesis of hybrid morpholino and DNA/RNA (modified/unmodified) oligonucleotides.
  • PMOs exhibit fast plasma clearance and poor cellular uptake.
  • PMOs can only be used to complement mRNA. PMOs cannot be used for the degradation of mRNA since they do not activate ribonuclease H1 binding to the substrate. Hence PMOs are not useful for degradation of STAT3 mRNA via an RNase H1 mechanism.
  • Caruthers et al. developed a novel method for the efficient and automatic synthesis of antisense oligonucleotide containing the morpholino nucleoside joined through 3’-thiophosphoramidate linkages (thiomorpholino oligonucleotides, TMO). This method enabled the synthesis of an antisense oligonucleotide with only thiomorpholino oligonucleotides or hybrid thiomorpholino and DNA/RNA (modified) linkages..
  • the hybrid morpholino and DNA/RNA(modified) antisense oligonucleotides can bind with Ribonuclease H and cause mRNA degradation whereas the all thiomorpholino oligonucleotide constructs can interact with complementary RNA/DNA and block the expression of a gene.
  • the antisense oligonucleotides described herein include novel antisense inhibitors for STAT3 containing the TMO structure motif, which are further designed to be complementary to the Signal Transducer and Activator of Transcription 3 (STAT3) mRNA sequence.
  • STAT3 Signal Transducer and Activator of Transcription 3
  • the ASOs of the invention are further configured to cause to the degradation of STAT3 mRNA, and the inhibition of STAT3 translation resulting in reduction of STAT3 protein.
  • the ASOs of the invention can further configured to induce alternative splicing of STAT3 resulting in an increase of the level of STAT3 ⁇ isoform compared to, or relative to the level of STAT3 ⁇ isoform in vivo or in vitro.
  • the novel TMOs of the invention offer different physiochemical properties, selectivity, and subcellular localization than traditional ASOs incorporating standard chemistry reported in the literature, and can exhibit different protein binding properties and nuclear localization, which can translate into different in vivo efficacy.
  • the present invention relates to a plurality of novel chimeric antisense oligonucleotides containing morpholine 3’-thiophosphoramidate nucleotides or morpholine 3’- thiophosphoramidate and DNA/RNA (modified) nucleotides having phosphorothioates (pS) linkers.
  • the ASOs of the invention are designed to be complementary to nucleic acid sequences within the Signal Transducer and Activator of Transcription 3 (STAT3) pre-mRNA or mature sequences, leading to the degradation of STAT3 mRNA or inhibition of STAT3 translation.
  • STAT3 Signal Transducer and Activator of Transcription 3
  • the chimeric ASOs of the invention are preferably selected from the group consisting of SEQ ID NO’s: 1-7, and 9-40, and combinations thereof. This includes sequences which can hybridize to such sequences under stringent hybridization conditions, sequences complementary thereto, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof which inhibit STAT3 (SEQ ID NO’s: 41-44).
  • the chimeric ASO oligomers of the invention further include a modified backbone structure and least a portion that is complementary to a target region of STAT3 mRNA, preferably selected from SEQ ID NO’s: 1-7, and 9-40, that induces alternative splicing of STAT3 resulting in an increase of STAT3 ⁇ isoform relative to STAT3 ⁇ isoform.
  • the chimeric ASOs of the invention can be used for the treatment of STAT3 pathway related diseases, which have been implicated in tumor initiation, progression, metastasis, drug resistance and immune suppression.
  • the novel chimeric ASOs of the invention can be used for the treatment of cancer or autoimmune diseases, preferably in humans, for example through the inhibition of STAT3, or the increase of STAT3 ⁇ isoform relative to STAT3 ⁇ isoform via ASO-mediated alternative splicing of STAT3, in a subject in need thereof. Additional aspect of the invention will be apparent from the figures, specification and claims provided below. BRIEF DESCRIPTION OF THE FIGURES FIG. 1: shows the STAT3 signal pathway and its regulation of exemplary cancer and immune-related genes; FIG.
  • FIG. 2A-B shows exemplary antisense oligonucleotides monomer, having various modifications including DNA/RNA(modified) phosphorothioate structure motifs;
  • FIG. 3 shows TMO chimeric ASOs cause a significant reduction in STAT3 mRNA levels in 293T cells and the inhibitory effect of a subset of chimeric ASOs is comparable to ISIS No.481464 at the same concentration.
  • FIG. 4 shows dose-dependent inhibition of human STAT3 in 293T cells demonstrating that TMOs are comparable in potency to suppression STAT3 mRNA.
  • FIG. 5 shows that TMO chimeric ASO can be administrated to cell lines without transfection reagent and free uptake of TMO chimeric ASO reduced the levels of STAT3 mRNA in a dose-dependent manner.
  • FIG 6A-B PCR results in splice switching demonstrating relative amount of STAT3 ⁇ decreased and STAT3 ⁇ increased compared with control after TMO treatment in 293T Cells and Hela Cells. (100nM or 500nM TMO, 4-day treatment). Dan: danvatirsen
  • FIG 7 Western Blot in Hela cells; 500nM, 4-days treatment.
  • FIG. 9A-B (A) shows RT-qPCR results on the inhibition of mANG (Reporter) and endogenous STAT3 in 293T-EP160 cell line using 50 nM ASO; (B) shows RT-qPCR results on the inhibition of mANG (Reporter) and endogenous STAT3 in 293T-EP160 cell line using 50 nM ASO.
  • FIG.10 shows ASO knockdown of STAT3 in H460 cells using 200 nM dose via gymnotic delivery compared with positive control danvatirsen and negative control, non-targeting KV66 (SEQ ID NO.8).
  • STAT3 related diseases or conditions such as cancer, autoimmune/immune diseases, or any disease or condition that may benefit from inhibiting expression of STAT3, by administering one or more antisense oligonucleotide compounds of the invention that are specifically designed to inhibit STAT3 protein expression.
  • these methods are useful in the prophylaxis and treatment of STAT3 related diseases or conditions.
  • the methods described herein further provide improved treatment options for patients with STAT3 related diseases or conditions and offer significant and practical advantages over alternate methods of inhibiting STAT3 protein expression.
  • the improved methods of this disclosure relate to the administration of an antisense compound for inhibiting STAT3 protein expression that may require lower doses and/or less frequent dosing regimens than prior approaches.
  • the present invention further provides improved methods and compositions for treating STAT3 related diseases or conditions, such as cancer, autoimmune/immune diseases, or any disease or condition that may benefit from modulating the expression/levels of STAT3 isoforms.
  • the invention includes the administration of one or more antisense oligonucleotide compounds that induce alternative splicing of STAT3 resulting in an increase of STAT3 ⁇ isoform relative to STAT3 ⁇ isoform.
  • these methods are useful in the prophylaxis and treatment of STAT3 related diseases or conditions where higher levels of isoform STAT3 ⁇ ⁇ have been demonstrated to have a therapeutic/anti-cancer effect.
  • the invention includes one or more novel antisense oligonucleotides containing morpholine 3-thiophosphoramidate nucleotides or morpholine 3-thiophosphoramidate and DNA/RNA (modified) nucleotides having phosphorothioates (pS) linkers. They are specifically designed to be complementary to nucleic acid sequences within the Signal Transducer and Activator of Transcription 3 (STAT3) pre-mRNA or mature sequences, leading to the degradation of STAT3 mRNA or inhibition of STAT3 translation. They are used for the treatment of diseases that could be modulated by the STAT3 pathway, which has been implicated in tumor initiation, progression, metastasis, drug resistance and immune suppression.
  • STAT3 Signal Transducer and Activator of Transcription 3
  • STAT3 means reducing expression of STAT3 mRNA and/or protein levels in the presence of a STAT3 ASO compound, including a STAT3 chimeric ASOs of the invention, as compared to expression of STAT3 mRNA and/or protein levels in the absence of a STAT3 antisense compound.
  • STAT3 signal transducer and activator of transcription 3 (acute-phase response),” and “signal transducer and activator of transcription 3” can be used interchangeably and refer to a transcription factor encoded by a gene designated in human as the STAT3 gene, which has a human gene map locus of 17q21 and described by Entrez Gene cytogenetic band: 17q21.31; Ensembl cytogenetic band: 17q21.2; and, HGNC cytogenetic band: 17q21.
  • STAT3 refers to a human protein that has 770 amino acids and has a molecular weight of about 88,068 Da.
  • STAT3 refers to any form of STAT3 known to those of skill in the art, including, but not limited to, STAT3 ⁇ (SEQ ID NO.45) and STAT3 ⁇ (SEQ ID NO: 46).
  • STAT3 mRNA or RNA sequences or sense strands means an STAT3 RNA isoform as set forth in SEQ ID NO;s: 41-46, as well as variants and homologs having at least 80% or more identity with human STAT3 mRNA sequence as set forth in any one of SEQ ID NO’s: 41-46.
  • the term “reduce” or “inhibit” may relate generally to the ability of one or more antisense compounds of the invention to “decrease” a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic art. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to persons skilled in the art, and may include reductions in the symptoms or pathology of in STAT3 related disease, or reductions in the expression of STAT3.
  • a “decrease” in a response may be statistically significant as compared to the response produced by no antisense compound or a control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in between.
  • the term “reduce” or “inhibit” may relate generally to the ability of one or more antisense compounds of the invention to target a STAT3 mRNA, resulting in a disease in the STAT3 protein, preferably in a cell, and even more preferably in a cancer call of a subject in need thereof.
  • the term “reduce” or “inhibit” can also relate generally to the ability of one or more antisense compounds of the invention to target a STAT3 mRNA and induce alternative splicing, preferably of exon 23, and thus increase STAT3 ⁇ isoform while reducing the STAT3 ⁇ isoform, preferably in a cell, and even more preferably in a cancer call of a subject in need thereof.
  • antisense compound comprises an antisense oligonucleotide (ASO), and preferably chimeric ASOs having morpholine 3’-thiophosphoramidate nucleotides or morpholine 3’-thiophosphoramidate and DNA/RNA (modified) nucleotides having phosphorothioates (pS) linkers.
  • ASO antisense oligonucleotide
  • chimeric ASOs having morpholine 3’-thiophosphoramidate nucleotides or morpholine 3’-thiophosphoramidate and DNA/RNA (modified) nucleotides having phosphorothioates (pS) linkers.
  • STAT3 expression is inhibited by administering a therapeutically effective amount of a ASO composition of the invention, and preferably a pharmaceutical composition containing a chimeric ASO having morpholine 3’- thiophosphoramidate nucleotides or morpholine 3’-thiophosphoramidate and DNA/RNA (modified) nucleotides having phosphorothioates (pS) linkers, that selectively binds to a complementary target sequence in STAT3 pre-mRNA.
  • a ASO composition of the invention and preferably a pharmaceutical composition containing a chimeric ASO having morpholine 3’- thiophosphoramidate nucleotides or morpholine 3’-thiophosphoramidate and DNA/RNA (modified) nucleotides having phosphorothioates (pS) linkers, that selectively binds to a complementary target sequence in STAT3 pre-mRNA.
  • an effective amount of an administered composition may comprise one of several amounts e.g., 2 mg/kg, about 5 mg/kg, about 10 mg/kg, or a dosage in the range of 15 mg/kg to 50 mg/kg, which includes a chimeric ASO as described herein, administered over a period of time sufficient to treat the disease or condition in a subject.
  • the ASOs of the invention are preferably selected from the group consisting of SEQ ID NO’s: 1-7, and 9-40, and combinations thereof. This includes sequences which can hybridize to such sequences under stringent hybridisation conditions, sequences complementary thereto, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof which possess or inhibit expression of STAT3.
  • Additional embodiments of the invention include chimeric ASO variants which include antisense oligomers having about or at least about 70% sequence identity or homology, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or homology, over the entire length of any of SEQ ID NO’s: 1-7, and 9-40.
  • RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or pairing such that stable and specific binding occurs between the oligomer and the DNA, cDNA, or RNA target. It is understood in the art that the sequence of an antisense oligomer need not be 100% complementary to that of its target sequence to be specifically hybridizable.
  • An antisense oligomer is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA product, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligomer to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
  • Selective hybridisation may be under low, moderate, or high stringency conditions, but is preferably under high stringency.
  • stringency of hybridisation will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands and the number of nucleotide base mismatches between the hybridising nucleic acids.
  • Stringent temperature conditions will generally include temperatures more than 30oC, typically in excess of 37oC, and preferably in excess of 45oC, preferably at least 50 ⁇ C, and typically 60 ⁇ C-80 ⁇ C or higher.
  • Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter.
  • the antisense oligomers of the present invention may include oligomers that selectively hybridize to the sequences, SEQ ID NO's: 1-7, and 9-40 as described herein.
  • the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide. Such hybridization may occur with “near” or “substantial” complementarity of the antisense oligomer to the target sequence, as well as with exact complementarity.
  • selective hybridisation will occur when there is at least about 55% identity over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75% and most preferably at least about 90%, 95%, 98% or 99% identity with the nucleotides of the antisense oligomer.
  • the length of homology comparison, as described, may be over longer stretches and in certain embodiments will often be over a stretch of at least about nine nucleotides, usually at least about 12 nucleotides, more usually at least about 20, often at least about 21, 22, 23 or 24 nucleotides, at least about 25, 26, 27 or 28 nucleotides, at least about 29, 30, 31 or 32 nucleotides, at least about 36 or more nucleotides.
  • the antisense oligomer sequences of the invention preferably have at least 75%, more preferably at least 85%, more preferably at least 86, 87, 88, 89 or 90% homology to the sequences shown in the sequence listings herein.
  • an antisense oligomer of the invention consists of less than about 30 nucleotides, it is preferred that the percentage identity is greater than 75%, preferably greater than 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96, 97, 98% or 99% compared with the antisense oligomers set out in the sequence listings herein.
  • Nucleotide homology comparisons may be conducted by sequence comparison programs such as the GCG Wisconsin Bestfit program or GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • the antisense oligomer of the present invention may have regions of reduced homology, and regions of exact homology with the target sequence. It is not necessary for an oligomer to have exact homology for its entire length.
  • the oligomer may have continuous stretches of at least 4 or 5 bases that are identical to the target sequence, preferably continuous stretches of at least 6 or 7 bases that are identical to the target sequence, more preferably continuous stretches of at least 8 or 9 bases that are identical to the target sequence.
  • the oligomer may have stretches of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 bases that are identical to the target sequence.
  • the remaining stretches of oligomer sequence may be intermittently identical with the target sequence; for example, the remaining sequence may have an identical base, followed by a non- identical base, followed by an identical base.
  • the oligomer sequence may have several stretches of identical sequence (for example 3, 4, 5 or 6 bases) interspersed with stretches of less than perfect homology. Such sequence mismatches will preferably have no or very little loss of cleavage modifying activity.
  • the invention includes one or more chimeric antisense oligonucleotide (ASO) containing at least one morpholino 3’-thiophosphoramidate nucleotides (TMO motif), and one or more DNA/RNA (modified) nucleosides having one or more 3’- phosphorothioate internucleotide linkages targeted to the STAT3 mRNA sequence and inhibit the expression of STAT3.
  • ASO chimeric antisense oligonucleotide
  • TMO motif morpholino 3’-thiophosphoramidate nucleotides
  • DNA/RNA (modified) nucleosides having one or more 3’- phosphorothioate internucleotide linkages targeted to the STAT3
  • the invention includes one or more chimeric antisense oligonucleotide (ASO) containing at least one morpholino 3’-thiophosphoramidate nucleotides (TMO motif), and one or more DNA/RNA (modified) nucleosides having one or more 3’- phosphorothioate internucleotide linkages targeted to exon 23 of the STAT3 mRNA sequence and induces alternative splicing of STAT3 resulting in an increase of STAT3 ⁇ isoform relative to STAT3 ⁇ isoform.
  • ASO chimeric antisense oligonucleotide
  • TMO motif morpholino 3’-thiophosphoramidate nucleotides
  • the invention may include a composition for inhibiting expression of Signal Transducer and Activator of Transcription 3 (STAT3) in a subject comprising an antisense oligonucleotide having at least 1 thiomorpholino nucleotide (TMO), which comprises a morpholino subunit, wherein the morpholino nitrogen of the morpholino subunit is linked by a thiophosphoramidate-containing internucleotide linkage to a 5’ exocyclic carbon of an adjacent nucleotide, or the 6’-exocyclic carbon of an adjacent morpholino subunit, or a TMO/DNA chimera, or a TMO/DNA/RNA (modified) chimera, and wherein at least a portion of said TMO is complementary to a target region of STAT3 mRNA.
  • TMO thiomorpholino nucleotide
  • the invention includes one or more isolated chimeric antisense oligonucleotide (ASO) having at least a portion that is complementary to a target region of a Signal Transducer and Activator of Transcription 3 (STAT3) mRNA, wherein the ASO is selected from the group consisting of: SEQ ID NO’s: 1 to 7, and 9 to 40, said sequence having a modified backbone structure, and wherein the antisense oligomer inhibits the expression of STAT3, or induces alternative splicing of STAT3 resulting in an increase of STAT3 ⁇ isoform relative to STAT3 ⁇ isoform.
  • ASO isolated chimeric antisense oligonucleotide
  • the ASO of the invention is between 5-and 50, and preferably between 10 to 30 monomers in length, and may further be configured to be complementary to a target region of STAT3 mRNA comprising intron 1, and/or exon 24 or 23.
  • one or more chimeric ASOs of the invention may include one or more morpholine 3’-thiophosphoramidate nucleotides, or one or more morpholine 3’- thiophosphoramidate nucleotides and DNA/RNA (modified) nucleotides, each having 3’- phosphorothioates internucleotide linkages.
  • one or more chimeric ASOs of the invention comprise a central gap segment having eight or ten 2’-deoxynucleosides and/or2’-deoxyribonucleosides, or a combination of the same, flanked on both sides by wings having three to six thiomorpholino oligonucleotides (TMOs), said TMOs having morpholino nucleosides joined through 3’-thiophosphoramidate linkages, and wherein the internucleotide linkages of the chimeric ASO further comprise phosphorothioate internucleotide linkages.
  • TMOs thiomorpholino oligonucleotides
  • one or more chimeric ASOs of the invention may be formed from monomer synthons selected from morpholine nucleoside 3’-phosphorodiamidites, DNA nucleoside 3’-phosphoramidites, or modified RNA nucleoside 3’-phosphoramidites, or a combination of the same.
  • the modified RNA nucleoside phosphoramidites are selected from the group of synthons consisting of 2’-O-methyl (2’OMe), 2’-O-methoxyethyl (2’- MOE); locked Nucleic Acid (LNA), ethylene-bridged nucleic acids (ENA), constrained ethyl nucleoside (2’-cEt), 2’-Flouro substituted, or a combination of the same.
  • one or more chimeric ASOs of the invention may further include nucleobases selected from the group consisting of: thymine, cytosine, adenine, guanine, uracil, 5’-methyl-cytosine, and pseudouridine.
  • one or more chimeric ASOs of the invention may be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.
  • one or more of the moieties or conjugates of the invention can be selected from the group consisting of: a targeting moiety, GalNAc conjugate, a peptide conjugate, an antibody conjugate, an anchoring moiety, and cholesterol.
  • one or more moieties or conjugates are connected to said ASO through a linker, which in a preferred embodiment can be selected from the group consisting of: cholesteryl TEG, and C6, among others know in the art.
  • Additional embodiments of the invention include method for inhibiting expression of Signal Transducer and Activator of Transcription 3 (STAT3) in a subject in need thereof, the method including the step of contacting one or more of the ASOs of the invention and allowing the oligomer(s) to bind to a target nucleic acid site, and preferably a target site on a STAT3 mRNA.
  • the method may include contacting one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 1 to 7, and 9 to 40.
  • Additional embodiments may include pharmaceutical compositions comprising one or more ASOs of the invention, and a pharmaceutically acceptable carrier.
  • a pharmaceutical compositions may preferably include one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 1 to 7, and 9 to 40.
  • Additional embodiments of the invention include method for inducing alternative splicing of STAT3 resulting in an increase of STAT3 ⁇ isoform relative to STAT3 ⁇ isoform in a subject in need thereof, the method including the step of contacting one or more of the ASOs of the invention and allowing the oligomer(s) to bind to a target nucleic acid site, and preferably exon 23 on a STAT3 mRNA.
  • the method may include contacting one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 22 to 36.
  • Additional embodiments may include pharmaceutical compositions comprising one or more ASOs of the invention, and a pharmaceutically acceptable carrier.
  • a pharmaceutical compositions may preferably include one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 22 to 36.
  • Additional embodiments include methods of treating, preventing or ameliorating the effects of a disease associated with STAT3, the method comprising the step of administering to a subject in need thereof, a therapeutically effective amount of one or more ASOs or a pharmaceutical compositions comprising one or more ASOs of the invention, and preferably one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 1 to 7, and 9 to 40.
  • a therapeutically effective amount of one or more ASOs or a pharmaceutical compositions comprising one or more ASOs of the invention, and preferably one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 1 to 7, and 9 to 40.
  • Additional embodiments include methods of treating, preventing or ameliorating the effects of a disease associated with STAT3, which is preferably cancer, or an autoimmune disease, the method comprising the step of administering to a subject in need thereof, a therapeutically effective amount of one or more ASOs or a pharmaceutical compositions comprising one or more ASOs of the invention, and preferably one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 1 to 7, and 9 to 40.
  • Additional embodiments include methods of treating, preventing or ameliorating the effects of a disease associated with STAT3, the method comprising the step of administering to a subject in need thereof, a therapeutically effective amount of one or more ASOs or a pharmaceutical compositions comprising one or more ASOs of the invention, and preferably one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 22 to 36, where the ASO induces alternative splicing of STAT3 resulting in an increase of STAT3 ⁇ isoform relative to STAT3 ⁇ isoform.
  • Additional embodiments include methods of treating, preventing or ameliorating the effects of a disease associated with STAT3, which is preferably cancer, or an autoimmune disease, the method comprising the step of administering to a subject in need thereof, a therapeutically effective amount of one or more ASOs or a pharmaceutical compositions comprising one or more ASOs of the invention, and preferably one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 22 to 36, wherein the ASO induces alternative splicing of the STAT3 resulting in an increase of STAT3 ⁇ isoform relative to STAT3 ⁇ isoform.
  • a disease associated with STAT3, which is preferably cancer, or an autoimmune disease the method comprising the step of administering to a subject in need thereof, a therapeutically effective amount of one or more ASOs or a pharmaceutical compositions comprising one or more ASOs of the invention, and preferably one or more of the ASOs selected from the group consisting of: SEQ ID NO’s: 22 to 36
  • the ASOs of the invention can be designed to block or inhibit translation of mRNA or to inhibit natural pre-mRNA splice processing and may be said to be “directed to” or “targeted against” a target sequence with which it hybridizes.
  • the target sequence is typically a region including an AUG start codon of an mRNA, a Translation Suppressing Oligomer, or splice site of a pre-processed mRNA, a Splice Suppressing Oligomer (SSO).
  • the target sequence for a splice site may include an mRNA sequence having its 5’ end 1 to about 25 base pairs downstream of a normal splice acceptor junction in a preprocessed mRNA.
  • An oligomer is more generally said to be “targeted against” a biologically relevant target, such as a protein, virus, or bacteria, when it is targeted against the nucleic acid of the target in the manner described above.
  • morpholino oligomer or “thiomorpholino oligomer” or “TMO” refer to an oligonucleotide analog composed of morpholino subunit structures (including thiomorpholinos), where (i) the structures are linked together by thiophosphoramidate containing linkages that join the morpholino nitrogen of one subunit to a 6’-exocyclic carbon of an adjacent morpholine subunit, or the 5’-exocyclic carbon of a nucleoside and (ii) each morpholino ring bears a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
  • the 5’ oxygen may be substituted with amino or lower alkyl substituted amino.
  • the pendant nitrogen attached to phosphorus may be unsubstituted, monosubstituted, or disubstituted with (optionally substituted) lower alkyl.
  • the purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine.
  • the antisense compounds can be prepared by stepwise solid-phase synthesis, employing methods detailed in PCT patent Application No. PCT/US17/51839, filed September 15, 2017, and further described below.
  • additional chemical moieties and conjugates to the antisense compound, e.g., to enhance pharmacokinetics or to facilitate capture or detection of the compound.
  • Such a moiety may be covalently attached, according to standard synthetic methods.
  • a moiety to enhance cellular uptake such as TAT, cholesteryl TEG, or an Fc binding immunoglobulin subunit, or a saccharide (such as a disaccharide, such as lactose), or a polyethylene glycol moiety or other hydrophilic polymer, e.g., one having 1-100 monomeric subunits, may be useful in enhancing solubility, increasing cellular uptake, or prolonging serum half-life, and the like.
  • a reporter moiety such as fluorescein, cyanine-5 or a radiolabeled group, may be attached for purposes of detection.
  • the reporter label attached to the oligomer may be a ligand, such as an antigen or biotin, capable of binding a labeled antibody or streptavidin.
  • a moiety for attachment or modification of an antisense compound it is desirable to select chemical compounds of groups that are biocompatible and likely to be tolerated by a subject without undesirable side effects.
  • a thiomorpholino (TMO) ring structure supports a base pairing moiety, to form a sequence of base pairing moieties which is typically designed to hybridize to a selected antisense target in a cell or in a subject being treated.
  • the base pairing moiety may be a purine or pyrimidine found in native DNA or RNA (e.g., the bases Adenine (A), Guanine (G), Cytosine (C), Thymine (T) or Uracil (U)) or an analog, such as hypoxanthine (the base component of the nucleoside inosine), 5-methyl cytosine, or 5-methyl uracil.
  • oligomers may have higher affinity for DNA and RNA than do the corresponding unmodified oligomers and demonstrate improved cell delivery, potency, and/or tissue distribution properties compared to oligomers having other internucleotide linkages.
  • the structural features and properties of the various linkage types and oligomers are described in more detail in the following discussion. The synthesis of these and related oligomers is described in PCT patent Application No. PCT/US17/51839, filed September 15, 2017, which is incorporated herein by reference in its entirety.
  • the antisense molecules used in these methods may be adapted to minimize or prevent cleavage by endogenous RNase H.
  • RNA-antisense oligonucleotide duplexes This property is highly preferred as the treatment of the RNA with the unmethylated oligonucleotides either intracellularly or in crude extracts that contain RNase H leads to degradation of the pre-mRNA-antisense oligonucleotide duplexes. Any form of modified antisense molecules that is capable of by-passing or not inducing such degradation may be used in the present methods.
  • antisense molecules which when duplexed with RNA, are not cleaved by cellular RNase H is 2’-O-methyl derivatives.2’-O-methyl-oligoribonucleotides are very stable in a cellular environment and in animal tissues, and their duplexes with RNA have higher Tm values than their ribo- or deoxyribo-counterparts. While antisense oligonucleotides are a preferred form of the antisense molecules, this disclosure comprehends other oligomeric antisense molecules, including but not limited to oligonucleotide mimetics.
  • oligonucleotides containing modified backbones or non-natural inter-nucleotide linkages include those that retain a phosphorothioate in the backbone and those that do not have a phosphorothioate in the backbone.
  • modified oligonucleotides that do not have a phosphorothioate in their inter-nucleoside backbone can also be considered to be oligonucleotides.
  • Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleo-bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C and are presently preferred base substitutions, even more particularly when combined with 2’-O-methoxyethyl sugar modifications.
  • oligonucleotides of this disclosure involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a saccharide, such as the disaccharide lactose, a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or
  • antisense compounds that are chimeric compounds.
  • Chimeric antisense compounds or “chimeras” are antisense molecules, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • oligonucleotides typically contain at least one region wherein the oligonucleotide is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and/or an additional region for increased binding affinity for the target nucleic acid.
  • the antisense oligonucleotides of this disclosure may include oligonucleotide moieties conjugated to a moiety or conjugate, such as a cell-penetrating peptide CPP, preferably an arginine- rich peptide transport moiety effective to enhance transport of the compound into cells.
  • the transport moiety is preferably attached to a terminus of the oligomer.
  • the peptides have the capability of inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells of a given cell culture population, including all integers in between, and allow macromolecular translocation within multiple tissues in vivo upon systemic administration.
  • the cell-penetrating peptide may be an arginine-rich peptide transporter.
  • the cell-penetrating peptide may be Penetratin or the TAT peptide.
  • a particularly preferred approach to conjugation of peptides to antisense oligonucleotides can be found in PCT publication No. WO2012/150960, which is incorporated herein by reference in its entirety.
  • a preferred embodiment of a peptide conjugated oligonucleotides of this disclosure utilizes glycine as the linker between the CPP and the antisense oligonucleotide.
  • a preferred peptide conjugated PMO consists of R6-G-TMO.
  • Uptake is preferably enhanced at least ten-fold, and more preferably twenty-fold, relative to the unconjugated compound.
  • arginine-rich peptide transporters i.e., cell-penetrating peptides
  • Certain peptide transporters have been shown to be highly effective at delivery of antisense compounds into primary cells including muscle cells (Marshall, Oda et al.2007; Jearawiriyapaisarn, Moulton et al.2008; Wu, Moulton et al.2008).
  • the antisense oligonucleotides of this disclosure may include oligonucleotide moieties conjugated to a CPP, preferably an arginine-rich peptide transport moiety effective to enhance transport of the compound into cells.
  • the transport moiety is preferably attached to a terminus of the oligomer.
  • the peptides have the capability of inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells of a given cell culture population, including all integers in between, and allow macromolecular translocation within multiple tissues in vivo upon systemic administration.
  • the cell-penetrating peptide may be an arginine-rich peptide transporter.
  • the cell-penetrating peptide may be Penetratin or the TAT peptide.
  • a particularly preferred approach to conjugation of peptides to antisense oligonucleotides can be found in PCT publication No. WO2012/150960, which is incorporated herein by reference in its entirety.
  • a preferred embodiment of a peptide conjugated oligonucleotides of this disclosure utilizes glycine as the linker between the CPP and the antisense oligonucleotide.
  • a preferred peptide conjugated PMO consists of R 6 -G-TMO.
  • Uptake is preferably enhanced at least ten-fold, and more preferably twenty-fold, relative to the unconjugated compound.
  • arginine-rich peptide transporters i.e., cell-penetrating peptides
  • Certain peptide transporters have been shown to be highly effective at delivery of antisense compounds into primary cells including muscle cells (Marshall, Oda et al.2007; Jearawiriyapaisarn, Moulton et al.2008; Wu, Moulton et al.2008).
  • the peptide transporters described herein when conjugated to an antisense TMO, demonstrate an enhanced ability to alter splicing of several gene transcripts (Marshall, Oda et al. 2007).
  • all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
  • “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by base-pairing rules. For example, the sequence “T-G-A (5’- 3’),” is complementary to the sequence “T-C-A (5’-3’).” Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to base pairing rules.
  • nucleic acids there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is often desired, some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches with respect to the target RNA. Variations at any location within the oligomer are included. In certain embodiments, variations in sequence near the termini of an oligomer are generally preferable to variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 nucleotides of the 5’ and/or 3’ terminus.
  • antisense oligomer and “antisense compound” and “antisense oligonucleotide” are used interchangeably and refer to a sequence of cyclic nucleotides, each bearing a base-pairing moiety, linked by internucleotide linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence.
  • the cyclic subunits are based on ribose or another pentose sugar or, in a preferred embodiment, a thiomorpholino group.
  • the oligomer may have exact or near sequence complementarity to the target sequence; variations in sequence near the termini of an oligomer are generally preferable to variations in the interior.
  • the cyclic subunits may be based on ribose or another pentose sugar or, in certain embodiments, a morpholino group (see description of morpholino oligonucleotides below).
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • 2’-OMe 2’-O-Methyl
  • the antisense oligonucleotides have the chemical composition of a naturally occurring nucleic acid molecule, i.e., the antisense oligonucleotides do not include a modified or substituted base, sugar, or inter-subunit linkage.
  • “antisense oligomer” and “antisense compound” and “antisense oligonucleotide” of the inventions includes one or more chimeric ASOs having morpholine 3’- thiophosphoramidate nucleotides or morpholine 3’-thiophosphoramidate and DNA/RNA (modified) nucleotides having phosphorothioates (pS) linkers.
  • the antisense oligonucleotides of the present invention are non- naturally occurring nucleic acid molecules, or “oligonucleotide analogues”.
  • non-natural nucleic acids can include one or more non-natural base, sugar, and/or inter- subunit linkage, e.g., a base, sugar, and/or linkage that has been modified or substituted with respect to that found in a naturally occurring nucleic acid molecule. Exemplary modifications are described below.
  • non-naturally occurring nucleic acids include more than one type of modification, e.g., sugar and base modifications, sugar and linkage modifications, base and linkage modifications, or base, sugar, and linkage modifications.
  • the antisense oligonucleotides contain a non-natural (e.g., modified or substituted) base.
  • the antisense oligonucleotides contain a non-natural (e.g., modified or substituted) sugar.
  • the antisense oligonucleotides contain a non-natural (e.g., modified or substituted) inter-subunit linkage.
  • the antisense oligonucleotides contain more than one type of modification or substitution, e.g., a non-natural base and/or a non- natural sugar, and/or a non-natural inter-subunit linkage.
  • non-naturally occurring antisense oligomers having (i) a modified backbone structure, e.g., a backbone other than the standard phosphodiester linkage found in naturally occurring oligo- and polynucleotides, and/or (ii) modified sugar moieties, e.g., morpholino moieties rather than ribose or deoxyribose moieties.
  • a modified backbone structure e.g., a backbone other than the standard phosphodiester linkage found in naturally occurring oligo- and polynucleotides
  • modified sugar moieties e.g., morpholino moieties rather than ribose or deoxyribose moieties.
  • Oligonucleotide analogues support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analogue backbone presents the bases in a manner to permit such hydrogen bonding in a sequence-specific fashion between the oligonucleotide analogue molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA).
  • Preferred analogues are those having a substantially uncharged, phosphorus containing backbone.
  • antisense oligomers One method for producing antisense oligomers is the methylation of the 2’ hydroxyribose position and the incorporation of a phosphorothioate backbone produces molecules that superficially resemble RNA but that are much more resistant to nuclease degradation, although persons skilled in the art of the invention will be aware of other forms of suitable backbones that may be useable in the objectives of the invention.
  • the antisense oligomers used in the method may be adapted to minimise or prevent cleavage by endogenous RNase H.
  • Antisense molecules that do not activate RNase H can be made in accordance with known techniques (see, e.g., U.S. Pat.
  • Such antisense molecules which may be deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modification which sterically hinders or prevents binding of RNase H to a duplex molecule containing the oligonucleotide as one member thereof, which structural modification does not substantially hinder or disrupt duplex formation. Because the portions of the oligonucleotide involved in duplex formation are substantially different from those portions involved in RNase H binding thereto, numerous antisense molecules that do not activate RNase H are available.
  • RNA:antisense oligomer duplexes any form of modified antisense oligomers that is capable of by-passing or not inducing such degradation may be used in the present method.
  • the nuclease resistance may be achieved by modifying the antisense oligomers of the invention so that it comprises partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups including carboxylic acid groups, ester groups, and alcohol groups.
  • antisense oligomers which when duplexed with RNA are not cleaved by cellular RNase H is 2’-O-methyl derivatives.
  • Such 2’-O-methyl-oligoribonucleotides are stable in a cellular environment and in animal tissues, and their duplexes with RNA have higher Tm values than their ribo- or deoxyribo-counterparts.
  • the nuclease resistant antisense oligomers of the invention may have at least one of the last 3’-terminus nucleotides fluoridated.
  • nuclease resistant antisense oligomers of the invention have phosphorothioate bonds linking between at least two of the last 3-terminus nucleotide bases, preferably having phosphorothioate bonds linking between the last four 3’-terminal nucleotide bases.
  • the antisense oligomer may be chosen from the list comprising: phosphoramidate or phosphorodiamidate morpholino oligomer (PMO); PMO-X; PPMO; peptide nucleic acid (PNA); a locked nucleic acid (LNA) and derivatives including alpha-L-LNA, 2’- amino LNA, 4’-methyl LNA and 4’-O-methyl LNA; ethylene bridged nucleic acids (ENA) and their derivatives; phosphorothioate oligomer; tricyclo-DNA oligomer (tcDNA); tricyclophosphorothioate oligomer; 2’O-Methyl-modified oligomer (2’-OMe); 2’-O-methoxy ethyl (2’-MOE); 2’-fluoro, 2’-fluroarabino (FANA); unlocked nucleic acid (UNA); hexitol nucleic acid (HNA); cyclohexohe
  • the abovementioned modified nucleotides are often conjugated with fatty acids, lipid, cholesterol, amino acids, carbohydrates, polysaccharides, or nanoparticles etc. to the sugar or nucleobase moieties.
  • conjugated nucleotide derivatives can also be used to construct antisense oligomers to modify cleavage factor binding.
  • Antisense oligomer-induced splicing factor binding modification of the STAT3 gene transcripts have generally used either oligoribonucleotides, PNAs, 2’OMe or MOE modified bases on a phosphorothioate backbone.
  • uracil (U) of the sequences provided herein may be replaced by a thymine (T).
  • such antisense molecules may be oligonucleotides wherein at least one, or all, of the inter-nucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates and phosphor amidates.
  • modified phosphates such as methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates and phosphor amidates.
  • every other one of the internucleotide bridging phosphate residues may be modified as described.
  • such antisense molecules are molecules wherein at least one, or all, of the nucleotides contain a 2’ lower alkyl moiety (e.g., Ci-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1- propenyl, 2-propenyl, and isopropyl).
  • a 2’ lower alkyl moiety e.g., Ci-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1- propenyl, 2-propenyl, and isopropyl.
  • every other one of the nucleotides may be modified as described.
  • Specific examples of antisense oligonucleotides useful in this invention include oligonucleotides containing modified backbones or non-natural inter-subunit linkages.
  • Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their inter-nucleoside backbone are also considered to be oligonucleosides. In other antisense molecules, both the sugar and the inter-nucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleo-bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • Oligonucleotides containing a modified or substituted base include oligonucleotides in which one or more purine or pyrimidine bases most commonly found in nucleic acids are replaced with less common or non-natural bases.
  • Purine bases comprise a pyrimidine ring fused to an imidazole ring; adenine and guanine are the two purine nucleobases most commonly found in nucleic acids. These may be substituted with other naturally occurring purines, including but not limited to N 6 -methyladenine, N 2 -methylguanine, hypoxanthine, and 7- methylguanine.
  • Pyrimidine bases comprise a six-membered pyrimidine ring; cytosine, uracil, and thymine are the pyrimidine bases most commonly found in nucleic acids. These may be substituted with other naturally occurring pyrimidines, including but not limited to 5-methylcytosine, 5- hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotides described herein contain thymine bases in place of uracil.
  • modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g.2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine (e.g.5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5- aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5- hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8- aza-7-deazaguanine, 8-aza- 7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguan
  • modified or substituted nucleo-bases are particularly useful for increasing the binding affinity of the antisense oligonucleotides of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C and are presently preferred base substitutions, even more particularly when combined with 2’-0-methoxyethyl sugar modifications. In some embodiments, modified or substituted nucleo-bases are useful for facilitating purification of antisense oligonucleotides.
  • antisense oligonucleotides may contain three or more (e.g., 3, 4, 5, 6 or more) consecutive guanine bases.
  • a string of three or more consecutive guanine bases can result in aggregation of the oligonucleotides, complicating purification.
  • one or more of the consecutive guanines can be substituted with inosine. The substitution of inosine for one or more guanines in a string of three or more consecutive guanine bases can reduce aggregation of the antisense oligonucleotide, thereby facilitating purification.
  • another modification of the antisense oligonucleotides involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl- 5- tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thioether
  • the antisense molecules used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
  • Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.).
  • One method for synthesising oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.
  • Another preferred chemistry is the phosphorodiamidate morpholino oligomer (PMO) oligomeric compounds, which are not degraded by any known nuclease or protease.
  • PMO phosphorodiamidate morpholino oligomer
  • Modified oligomers may also contain one or more substituted sugar moieties. Oligomers may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. Certain nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • At least one pyrimidine base of the oligonucleotide comprises a 5-substituted pyrimidine base, wherein the pyrimidine base is selected from the group consisting of cytosine, thymine, and uracil.
  • the 5- substituted pyrimidine base is 5-methylcytosine.
  • at least one purine base of the oligonucleotide comprises an N-2, N-6 substituted purine base.
  • the N- 2, N-6 substituted purine base is 2, 6-diaminopurine.
  • the antisense oligonucleotide includes one or more 5-methylcytosine substitutions alone or in combination with another modification, such as 2’-O-methoxyethyl sugar modifications.
  • the antisense oligonucleotide includes one or more 2, 6-diaminopurine substitutions alone or in combination with another modification.
  • the antisense oligonucleotide is chemically linked to one or more moieties, such as a polyethylene glycol moiety, or conjugates, such as an arginine-rich cell penetrating peptide that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.
  • the arginine-rich polypeptide is covalently coupled at its N-terminal or C-terminal residue to the 3’ or 5’ end of the antisense compound.
  • the antisense compound is composed of morpholino subunits and phosphorus-containing inter-subunit linkages joining a morpholino nitrogen of one subunit to a 5’ exocyclic carbon of an adjacent subunit.
  • Another modification of the oligomers of the invention involves chemically linking to the oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligomer.
  • Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl- rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H- phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, myristyl, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid,
  • the present invention also includes antisense oligomers that are chimeric compounds.
  • “chimeric” antisense oligomers or “chimeras,” in the context of this invention are antisense oligomers, particularly oligomers, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligomer compound.
  • oligomers typically contain at least one region wherein the oligomer is modified to confer upon the oligomer or antisense oligomer increased resistance to nuclease degradation, increased cellular uptake, and an additional region for increased binding affinity for the target nucleic acid.
  • An “exon” refers to a defined section of nucleic acid that encodes a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after either portions of a pre-processed (or precursor) RNA have been removed by splicing.
  • the mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA or tRNA.
  • an "intron” refers to a nucleic acid region (within a gene) that is not translated into a protein.
  • An intron is a non-coding section that is transcribed into a precursor mRNA (pre-mRNA), and subsequently removed by splicing during formation of the mature RNA.
  • An “effective amount” or “therapeutically effective amount” refers to an amount of therapeutic compound, such as an antisense oligonucleotide, administered to a human subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect. For an antisense oligonucleotide, this effect is typically brought about by inhibiting translation or natural splice-processing of a selected target sequence.
  • an effective amount may be variable such as 5 mg/kg of a composition comprising a thiomorpholino antisense oligonucleotide for a period of time to treat the subject.
  • the term “subject” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the subject of the herein disclosed methods can be a human, non- human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the subject is a mammal.
  • a patient refers to a subject afflicted with a disease or disorder.
  • the subject has been diagnosed with a need for inhibition or negative modulation of STAT3 prior to the administering step.
  • the subject has been diagnosed with a need for treatment of one or more oncological disorders or cancers associated with STAT3 dysfunction prior to the administering step.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • active treatment that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder
  • causal treatment that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease.
  • the subject is a mammal such as a primate, and, in a further aspect, the subject is a human.
  • subject also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • livestock e.g., cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.
  • diagnosisd means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.
  • diagnosis with a disorder treatable by STAT3 inhibition means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by a compound or composition that can inhibit or negatively modulate STAT3.
  • diagnosis with a need for inhibition of STAT3 refers to having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition characterized by a dysfunction in STAT3 activity.
  • a diagnosis can be in reference to a disorder, such as an oncological disorder or disease, cancer and/or disorder of uncontrolled cellular proliferation and the like, as discussed herein.
  • the term “diagnosed with a need for inhibition of STAT3 activity” refers to having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by inhibition of STAT3 activity.
  • “diagnosed with a need for modulation of STAT3 activity” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by modulation of STAT3 activity, e.g., negative modulation.
  • “diagnosed with a need for treatment of one or more disorder of uncontrolled cellular proliferation associated with STAT3 dysfunction” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have one or disorders of uncontrolled cellular proliferation, e.g., a cancer, associated with STAT3 dysfunction.
  • the phrase “identified to be in need of treatment for a disorder,” or the like, refers to selection of a subject based upon need for treatment of the disorder.
  • a subject can be identified as having a need for treatment of a disorder (e.g., a disorder related to STAT3 activity) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the disorder.
  • the identification can, in one aspect, be performed by a person different from the person making the diagnosis.
  • the administration can be performed by one who subsequently performed the administration.
  • the terms “administering”, and “administration” refer to any method of providing a pharmaceutical preparation to a subject.
  • Such methods include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
  • contacting refers to bringing a disclosed compound and a cell, target STAT3 protein, or other biological entity together in such a manner that the compound can affect the activity of the target (e.g., spliceosome, cell, etc.), either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent.
  • isolated means material that is substantially or essentially free from components that normally accompany it in its native state.
  • an “isolated polynucleotide,” as used herein, may refer to a polynucleotide that has been purified or removed from the sequences that flank it in a naturally-occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
  • TMOs thiomorpholino oligonucleotides
  • TMO/DNA thiomorpholino/DNA chimeras
  • All syntheses were carried out on 1 ⁇ mol scale using a 5’-O-DMT-2’-deoxyribonucleoside joined to a CPG solid support via a succinate linkage. All solid phase syntheses were carried out as DMT ON.
  • TMOs and TMO/DNA chimeras For the synthesis of TMOs and TMO/DNA chimeras, 0.1M appropriately protected morpholino 3’-phosphordiamidites and appropriately protected 2’-deoxyribonucleoside 3’-phosphoramidites were dissolved in anhydrous acetonitrile and the detritylations were achieved by using 3% solution of trichloroacetic acid in dichloromethane. A coupling time of 30 sec was used for the condensation reactions. Following condensation, sulfurization and capping were carried out.
  • cholesteryl-TEG Phosphoramidite (1- Dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2- cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite).
  • Cyanine 5 labelling on the 3’ end of the TMO/DNA chimeras, KKV78 and KKV79 was attained by cyanine 5 CPG (1-[3-(4- monomethoxytrityloxy)propyl]-3,3,3’,3’-tetramethylindodicarbocyanine chloride-1’-propyl-3-O- succinoyl-long chain alkylamino-CPG).
  • Cleavage of these 5’-protected DMT-on oligonucleotides from the solid support and deprotection of base and phosphorus protecting groups was carried out in 0.5 ml of 28 % aqueous ammonia at 55 ⁇ C for 16h.
  • exemplary synthesized ASOs were synthesized and screened for activity.
  • Cell culture and Antisense Compound Treatment The effects of antisense compounds on the level of STAT3 mRNA in human cells can be tested in vitro using a variety of tumor cell lines or primary cell lines in culture.
  • the established cell lines were purchased from American Type Culture Collection (ATCC) and grown on culture dishes following manufacturer’s guidelines.
  • Illustrative cell lines include but not limited to 293T, A431, MM.1R, NCI-H460, HepG2, Hep3B etc. These cell lines are typically maintained in 5% CO 2 37 o C incubator.
  • Cells can be seeded on 6 well or 48 well plates and treated with ASOs when the plated cells reach 75% confluency.
  • Antisense oligonucleotides can be introduced into culture cells using either cation ion lipid transfection reagents such as LIPOFECTAMIN 2000, Poly(ethyleneimine) (PEI) or RNAiMax (ThermoFisher).
  • antisense oligonucleotides are administered to cell lines through gymnotic delivery, i.e., free uptake without the aid of a transfection agent or electroporation.
  • ASO is mixed with PEI or LIPOFECTAMIN 2000 in OPTI-MEM serum-free medium (ThermoFisher) to achieve the desired concentration of antisense oligonucleotide.
  • the lipid concentration for ASO testing may range from a ratio of 1:3 to 1:10 per ug of ASO: per ug of PEI or Lipofectamine 2000.
  • ASO is mixed with lipids at room temperature for at least 15 min before adding to cell culture media.
  • RNA isolation Methods of RNA isolation are well known in the art.
  • Total RNA is prepared using the TRIZOL Reagent (ThermoFisher) according to the manufacturer’s recommended protocols. The amount of RNA harvested is measured by a Nanodrop device. Measurement of STAT3 mRNA Levels: The levels of STAT3 mRNA expression in mammalian cells can be measured in a variety of ways known in the art. Quantitative real-time PCR is a popular method of choice in the art. Quantitation of STAT3 mRNA may be accomplished by qPCR using the Bio-rad CFX Opus 384 Real-Time PCR System (Bio-rad, Richmond, California) following manufacturer’s instructions. TBP mRNA can be used as the reference gene as its expression rarely changes under a variety of experimental conditions.
  • RNA from control and treated samples is reverse transcribed to produce complementary DNA (cDNA) using a reverse transcriptase (RT).
  • RT reverse transcriptase
  • the cDNA is then used as templates for real-time PCR amplification.
  • the RT and real-time PCR reagents can be purchased from vendors, including Bio-rad, New England Biolabs, and ThermoFisher.
  • Primer sets used for amplifying STAT3 include: Forward 5’-CAGTTTCTGGCCCCTTGGAT-3’ (SEQ ID NO: 47) Reverse 5’-AAGCGGCTATACTGCTGGTC-3’ (SEQ ID NO: 48) and TBP (Forward 5’-CACGAACCACGGCACTGATT-3’ (SEQ ID NO: 49) Reverse 5’-TTTTCTTGCTGCCAGTCTGGAC-3’ (SEQ ID NO: 50) These primers were mixed with 2X qPCR mix containing SYBR® Green I, a double- stranded DNA (dsDNA) binding dye, to measure DNA amplification during each cycle of a PCR.
  • dsDNA double- stranded DNA
  • a quantification cycle or Cq value
  • Cq values can be used to evaluate the relative target abundance between two or more samples.
  • Example 2 Antisense inhibition of human STAT3 in 293T cells TMOs were designed as chimeric ASO targeting either a human STAT3 intron 1 present in pre-mRNA or a human STAT3 exon 24 (3’-untranslated region). Their effects on human STAT3 mRNA levels in cells were tested in 293T cells using the transfection reagent PEI or in NCI-H460 cells by gymnotic delivery.
  • the chimeric ASO presented in Table 2 were designed as chimeric ASO with either eight or ten 2’-deoxynucleosides in the central gap segment and are flanked on both sides by wings comprising three to six TMOs.
  • the inhibition efficiency of chimeric ASO on STAT3 was compared to a non-targeting control KKV66 or ISIS No.481464 which is a 3-10-3 cEt gapmer targeting human STAT3. In all experiments, a mock control with transfection reagent PEI alone was also included.
  • the STAT3 mRNA levels were quantified by quantitative real-time PCR analysis using a SYBR® Green based method to determine Cq value of each sample relative to reference TBP mRNA.
  • Two chimeric ASOs that exhibited significant inhibition of STAT3 along with the non- targeting control were further tested at various doses in NCI-H460 lung cancer cells via gymnotic delivery without a transfection reagent.
  • Cells were seeded on a 48 well plate overnight at a density of 40,000 per well in RPMI medium.
  • Chimeric ASOs were diluted in RPMI medium at the final concentration of 5 uM, 2.5 uM, 0.5 uM, 0.1 uM and 0.02 uM. Diluted ASOs were added dropwise to the culture wells. Forty-eight hours after treatment, media were removed, and cells were harvested by adding 200 ul Trizol reagent.
  • RNA was isolated and quantified by a nanodrop UV-VIS device (DeNovix).
  • Complementary cDNA was prepared, and real-time PCR analysis was performed as described in the method section.
  • the results are presented in Figure 5 and indicate that TMO chimeric ASOs can be administrated to cell lines without transfection reagent and free uptake of TMO chimeric ASO reduced the levels of STAT3 mRNA in a dose-dependent manner suggesting these ASOs can effectively penetrate the cell membrane.
  • Example 4 Targeting STAT3 Exon 23 for splicing switching or selective inhibition of STAT3 ⁇ isoform.
  • Applicants disclose a series of novel antisense oligonucleotide that affect alternative splicing of exon 23, and thus increase STAT3 ⁇ isoform while reducing the STAT3 ⁇ isoform.
  • Applicants have also designed antisense oligonucleotide that target Exon 23, causing degradation of the STAT3 ⁇ isoform only. This targeting isoform specific STAT3 strategy could be superior to pan-STAT3 inhibition.
  • Splice Switching STAT3 ⁇ to STAT3 ⁇ (Exon 23) 1 x 10 6 cells were lysed using 1mL TRIzol reagent (Thermo Fisher), and RNA was extracted according to the manufacturer’s instructions.
  • PCR reactions were amplified with an initial denaturing step of 95°C for 3 minutes, 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension step of 72°C for 5 minutes.
  • PCR reactions were mixed with 5 ⁇ L 6X purple gel loading dye (New England BioLabs) and 8 ⁇ L product was run on a 3% gel made with 0.5X TAE buffer and ethidium bromide. The gel was run at 110V until separation of the two PCR products was seen.
  • the samples were boiled at ⁇ 100°C for 5 minutes.4 ⁇ L Spectra Multicolor Broad Range Protein Ladder (Thermo Fisher) and 20 ⁇ L of each sample was loaded into a 1.0mm, 10-well, 8% SDS-PAGE gel. The gel was run at 100V until the dye front reached the end of the gel. Semi-dry transfer was used to transfer the protein from the gel to a 0.45 ⁇ m NC membrane (Cytiva). The transfer was run at 14V for 1.5 hours, and ponceau stain was used to confirm a successful transfer. The membrane was cut above 50kD to separate actin (45kD) and STAT3 (85kD). The membrane was blocked in 5% milk in TBST for 1 hour at room temperature.
  • the membrane was washed 4 times for 5 minutes in TBST on a shaker.4mL 1% BSA containing 1:1000 primary antibody was added to each section of the membrane and incubated overnight at 4°C on a shaker. The membrane was washed again as before, and 4mL 1% BSA containing 1:3000 secondary antibody was added to the membrane. Following 1 hour of incubation at room temperature, the membrane was washed again. Finally, the membrane was briefly dried and imaged via chemiluminescence using SuperSignal substrate (Thermo Fisher) in the Amersham ImageQuant 800 (Cytiva).
  • Table 1 Chemical steps used in the automated synthesizer for the preparation of regular DNA Reactions Reagents/solvents Time (seconds) Detrit lation 3% trichloroacetic acid in dichloromethane Flow 50 Sec
  • Table 2 Chemical steps used in the automated synthesizer for the preparation of fully modified TMOs Reactions Reagents/solvents Time (seconds)
  • Table 3 Chemical steps used in the automated synthesizer for the preparation of TMO/DNA chimeras Reactions Reagents/solvents Time (seconds) Detritylation 3% trichloroacetic acid in dichloromethane Flow 50 Sec SEQ Calculated Observed ID.
  • Table 4B TMO Chimeric ASOs targeting STAT3 Exon23 SEQ Calculated Observed ID.
  • TMO and TMO/DNA chimera sequences Upper case, underline Letters in the ASO Sequence: morpholine 3’-thiophosphoramidate; lower case letters in the ASO sequence: 2’-deoxynucleoside 3’-thiophosphate and 2’-deoxyribonucleoside at the 3’-end.
  • Table 4C The synthesis of TMO Chimeric ASOs including chemical modified RNA in the sequence. SEQ Calculated Observed ID.
  • TMO and TMO/DNA chimera sequences e letters in the ASO sequence: 2’-deoxynucleoside 3’-thiophosphate and 2’-deoxyribonucleoside at the 3’-end; upper case 2’-OMe : 2’-OMe-nucleoside 3’-thiophosphate and 2’-OMe-ribonucleoside at the 3’- end: upper case 2’-MOE : 2’-O-methoxyethyl-nucleoside 3’-thiophosphate and 2’- O-methoxyethyl- ribonucleoside at the 3’-end; upper case LNA : locked nucleic acid (LNA)
  • Myeloid ERK5 deficiency suppresses tumor growth by blocking protumor macrophage polarization via STAT3 inhibition.
  • AZD9150 a next-generation antisense oligonucleotide inhibitor of STAT3 with early evidence of clinical activity in lymphoma and lung cancer. Sci Transl Med 7, 314ra185 (2015). 11. Wong, A. L. A., Hirpara, J. L., Pervaiz, S., Eu, J.-Q., Sethi, G. & Goh, B.-C. Do STAT3 inhibitors have potential in the future for cancer therapy? Expert Opin Investig Drugs 26, 883–887 (2017). 12. Sen, M., Thomas, S. M., Kim, S., Yeh, J. I., Ferris, R. L., Johnson, J.

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Abstract

La présente invention concerne une série d'oligonucléotides antisens contenant des nucléotides de morpholine 3-thiophosphoramidate ou des nucléotides de morpholine 3-thiophosphoramidate et d'ADN/ARN (modifiés) ayant des lieurs de phosphorothioates (pS). Ils sont spécifiquement conçus pour être complémentaires aux séquences d'acide nucléique dans les séquences pré-ARNm ou matures du transducteur de signal et de l'activateur de transcription 3 (STAT3), conduisant à la dégradation de l'ARNm STAT3 ou à l'inhibition de la traduction de STAT3, ou induisant un épissage alternatif de STAT3 conduisant à une augmentation de l'isoforme STAT3β par rapport à l'isoforme STAT3α. Les oligonucléotides antisens sont utilisés pour le traitement de maladies qui pourraient être modulées par la voie STAT3, qui a été impliquée dans l'initiation de la tumeur, la progression, la métastase, la pharmacorésistance et la suppression immunitaire. Par conséquent, ces nouveaux oligonucléotides antisens peuvent être utilisés pour le traitement de maladies associées à STAT3, telles que le cancer ou des maladies auto-immunes.
PCT/US2023/077027 2022-10-14 2023-10-16 Nouveaux oligonucléotides antisens contenant un morpholino hybride et un adn/arn (modifié) avec un lieur phosphorothioate (ps) pour le traitement du cancer et de troubles auto-immuns WO2024081970A2 (fr)

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