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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
• 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.
• 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.
• 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.
• 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 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.
• 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, 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.
• 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.
• 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.
• 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.
• 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 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.
• 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.
• 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.
• 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 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)

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