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WO2008109494A1 - Composés d'acides nucléiques conçus pour inhiber l'expression du gène stat3 et utilisations de ceux-ci - Google Patents

Composés d'acides nucléiques conçus pour inhiber l'expression du gène stat3 et utilisations de ceux-ci Download PDF

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WO2008109494A1
WO2008109494A1 PCT/US2008/055606 US2008055606W WO2008109494A1 WO 2008109494 A1 WO2008109494 A1 WO 2008109494A1 US 2008055606 W US2008055606 W US 2008055606W WO 2008109494 A1 WO2008109494 A1 WO 2008109494A1
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strand
dsrna
nucleotides
mdrna
molecule
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PCT/US2008/055606
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WO2008109494B1 (fr
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Steven C. Quay
James Mcswiggen
Narendra K. Vaish
Mohammad Ahmadian
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Mdrna, Inc.
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Publication of WO2008109494A1 publication Critical patent/WO2008109494A1/fr
Publication of WO2008109494B1 publication Critical patent/WO2008109494B1/fr
Priority to US12/552,082 priority Critical patent/US20100105134A1/en
Priority to US13/327,545 priority patent/US20130011922A1/en

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    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present disclosure relates generally to compounds for use in treating one or more hyperproliferative disorders, for example leukemia, lymphoma, breast cancer, prostate cancer, multiple myeloma, and head and neck cancer, as well as tumor proliferation by gene silencing and, more specifically, to a nicked or gapped double-stranded RNA (dsRNA) comprising at least three strands that decreases expression of a signal transducer and activator of transcription 3 (STAT3) gene, and to uses of such dsRNA to treat or prevent one or more hyperproliferative disorders, for example leukemia, lymphoma, breast cancer, prostate cancer, multiple myeloma, and head and neck cancer, as well as tumor proliferation associated with inappropriate STAT3 gene expression.
  • the dsRNA that decreases STAT3 gene expression may optionally have at least one uridine substituted with a 5-methyluridine.
  • RNA interference refers to the cellular process of sequence specific, post-transcriptional gene silencing in animals mediated by small inhibitory nucleic acid molecules, such as a double-stranded RNA (dsRNA) that is homologous to a portion of a targeted messenger RNA (Fire et al., Nature 391:806, 1998; Hamilton et al., Science 286:950, 1999).
  • dsRNA double-stranded RNA
  • RNAi has been observed in a variety of organisms, including mammalians (Fire et al., Nature 391:806, 1998; Bahramian and Zarbl, MoI. Cell. Biol. 19:21 A, 1999; Wianny and Goetz, Nature Cell Biol. 2:10, 1999).
  • RNAi can be induced by introducing an exogenous 21-nucleotide RNA duplex into cultured mammalian cells (Elbashir et al., Nature 411:494, 2001a).
  • the mechanism by which dsRNA mediates targeted gene-silencing can be described as involving two steps.
  • the first step involves degradation of long dsRNAs by a ribonuclease III- like enzyme, referred to as Dicer, into short interfering RNAs (siRNAs) having from 21 to 23 nucleotides with double-stranded regions of about 19 base pairs and a two nucleotide, generally, overhang at each 3'-end (Berstein et al., Nature 409:363, 2001; Elbashir et al., Genes Dev. 75:188, 2001b; and Kim et al, Nature Biotech. 23:222, 2005).
  • siRNAs short interfering RNAs
  • RNAi gene-silencing involves activation of a multi-component nuclease having one strand (guide or antisense strand) from the siRNA and an Argonaute protein to form an RNA-induced silencing complex ("RISC") (Elbashir et al., Genes Dev. 75:188, 2001).
  • RISC RNA-induced silencing complex
  • Argonaute initially associates with a double-stranded siRNA and then endonucleolytically cleaves the non-incorporated strand (passenger or sense strand) to facilitate its release due to resulting thermodynamic instability of the cleaved duplex (Leuschner et al., EMBO 7:314, 2006).
  • the guide strand is now able to bind a complementary target mRNA and the activated RISC cleaves the mRNA to promote gene silencing. Cleavage of the target RNA occurs in the middle of the target region that is complementary to the guide strand (Elbashir et al., 2001b).
  • Transcription factors encoded by members of the signal transducer and activator of transcription 3 (STAT3) family are involved in the survival, proliferation, differentiation, and apoptosis of cells (Kusmartsev, et al, Cancer Metastasis Rev. 25:3, 2006).
  • the STAT3 subfamily comprises at least three human isoforms: STAT3- ⁇ , STAT3- ⁇ , and STAT3- ⁇ .
  • STAT3- ⁇ and STAT3- ⁇ are both trunctated versions of STAT3- ⁇ (Yu et al., J. Am. Soc. Nephrol. 75:585, 2004; Kato et al., J. Biol. Chem. 279:30, 2004).
  • Each of the isoforms includes a STAT src homology 2 (SH2) domain (Yu et al., 2004).
  • STAT activation is dependent upon tyrosine phosphorylation of receptors on target tyrosine residues, which induce rearrangement and phosphotyrosine-SH2 dimerization between two STAT3s (Pedranzini et al., J. Clin. Invest. 114:5, 2004).
  • Phosphorylation of STAT3 can be from many different tyrosine kinases, including Janus kinases (JAKs), receptor tyrosine kinases (RTKs), and non-RTKs (Pedranzini et al., J. Clin. Invest. 114:5, 2004).
  • JJAKs Janus kinases
  • RTKs receptor tyrosine kinases
  • non-RTKs Pedranzini et al., J. Clin. Invest. 114:5, 2004.
  • activated STAT3 has been shown in most tumors and is crucial for tumor-cell proliferation and survival (Kusmartsev, et al., 2006; Heck et al., J. Virol. 79:9, 2005).
  • overexpression of activated STAT3 has been found in leukemias and lymphomas, such as large granular lymphocyte leukemia and non-Hodgkins lymphoma, as well as solid tumors, such as breast and prostate cancer (Secko, CMAJ 173:3, 2005).
  • STAT3 is persistently tyrosine phosphorylated either as a consequence of deregulated positive effectors of STAT activation, such as tyrosine kinases, or negative regulators of STAT phosphorylation, for example, phosphatases, suppressor of cytokine signaling, protein inhibitor of activated STATs (Pedranzini et al., 2004). Additionally, continual activation of STAT3 has been found to be necessary for cell growth in multiple myeloma and head and neck cancer (Secko, 2005).
  • dsRNA nicked or gapped double-stranded RNA
  • STAT3 signal transducer and activator of transcription 3
  • mRNA messenger RNA
  • the first strand is about 15 to about 40 nucleotides in length and is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence that is complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides of any one of STAT3 mRNA as set forth in SEQ ID NO: 1158, 1159, or 1160.
  • the mdRNA is a RISC activator (e.g., the first strand has about 15 nucleotides to about 25 nucleotides) or a Dicer substrate (e.g. , the first strand has about 26 nucleotides to about 40 nucleotides).
  • the gap comprises at least one to ten unpaired nucleotides in the first strand positioned between the double-stranded regions formed by the second and third strands when annealed to the first strand, or the gap is a nick.
  • the nick or gap is located 10 nucleotides from the 5'-end of the first (antisense) strand or at the Argonaute cleavage site.
  • the meroduplex nick or gap is positioned such that the thermal stability is maximized for the first and second strand duplex and for the first and third strand duplex as compared to the thermal stability of such meroduplexes having a nick or gap in a different position.
  • the instant disclosure provides an mdRNA molecule having a first strand that is complementary to human STAT3 mRNA as set forth in SEQ ID NO:1158, 1159, or 1160, and a second strand and a third strand that is each complementary to non-overlapping regions of the first strand, wherein the second strand and third strand can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein (a) the mdRNA molecule optionally includes at least one double-stranded region comprises from 5 base pairs to 13 base pairs, or (b) the double-stranded regions combined total about 15 base pairs to about 40 base pairs and the mdRNA molecule optionally has blunt ends; and wherein at least one pyrimidine of the mdRNA comprises a pyrimidine nucleoside according to Formula I or II:
  • R 1 and R 2 are each independently a -H, -OH, -OCH 3 , -OCH 2 OCH 2 CH 3 ,
  • At least one R 2 is selected from 2'-0-(Ci-Cs) alkyl, 2'-O-methyl, 2'-OCH 2 OCH 2 CH 3 , 2'-OCH 2 CH 2 OCH 3 , 2'-0-allyl, or fluoro.
  • at least one pyrimidine nucleoside of the mdRNA molecule is a locked nucleic acid (LNA) in the form of a bicyclic sugar, wherein R 2 is oxygen, and the 2'-0 and 4'-C form an oxymethylene bridge on the same ribose ring (e.g., a 5-methyluridine LNA) or is a G clamp.
  • LNA locked nucleic acid
  • one or more of the nucleosides are according to Formula I in which R 1 is methyl and R 2 is a 2'-0-(Ci-Cs) alkyl, such as 2'-0-methyl.
  • the gap comprises at least one unpaired nucleotide in the first strand positioned between the double- stranded regions formed by the second and third strands when annealed to the first strand, or the gap is a nick.
  • the nick or gap is located 10 nucleotides from the 5 '-end of the first strand or at the Argonaute cleavage site.
  • some embodiments provide an mdRNA molecule having a 5-methyluridine (ribothymidine) or a 2-thioribothymidine in place of at least one uridine on the first, second, or third strand, or in place of each and every uridine on the first, second, or third strand.
  • the mdRNA further comprises one or more non-standard nucleoside, such as a deoxyuridine, locked nucleic acid (LNA) molecule, or a universal-binding nucleotide, or a G clamp.
  • some embodiments provide an mdRNA comprising an overhang of one to four nucleotides on at least one 3 '-end that is not part of the gap, such as at least one deoxyribonucleotide or two deoxyribonucleotides (e.g., thymidine).
  • at least one or two 5'-terminal ribonucleotide of the second strand within the double-stranded region comprises a 2'-sugar substitution.
  • at least one or two 5 '-terminal ribonucleotide of the first strand within the double-stranded region comprises a 2'-sugar substitution.
  • Figure 1 shows the average gene silencing activity of intact (first bar), nicked (middle bar) and gapped (last bar) dsRNA Dicer substrate specific for each of 22 different targets (AKT, EGFR, FLTl, FRAPl, HIFlA, IL17A, IL18, IL6, MAP2K1, MAPKl, MAPK14, PDGFA, PDGFRA, PIKC3A, PKN3, RAFl, SRD5A1, TNF, TNFSF13B, VEGFA, BCR-ABL [b2a2], and BCR-ABL [b3a2]).
  • Each bar is a graphical representation of an average activity often different sequences for each target, which is calculated from the data found in Table 1.
  • Figure 2 shows knockdown activity for RISC activator lacZ dsRNA (21 nucleotide sense strand/21 nucleotide antisense strand; 21/21), Dicer substrate lacZ dsRNA (25 nucleotide sense strand/27 nucleotide antisense strand; 25/27), and meroduplex lacZ mdRNA (13 nucleotide sense strand and 11 nucleotide sense strand/27 nucleotide antisense strand; 13, 11/27 - the sense strand is missing one nucleotide so that a single nucleotide gap is left between the 13 nucleotide and 11 nucleotide sense strands when annealed to the 27 nucleotide antisense strand.
  • G 1498 single stranded 21 nucleotide antisense strand alone (designated AS-only) was used as a control.
  • Figure 4 shows knockdown activity of a lacZ dicer substrate (25/27) having a nick in one of each of positions 8 to 14 and a one nucleotide gap at position 13 of the sense strand (counted from the 5 '-end).
  • a dideoxy guanosine (ddG) was incorporated at the 5 '-end of the 3 '-most strand of the nicked or gapped sense sequence at position 13.
  • Figure 7 shows knockdown activity of a dicer substrate influenza dsRNA Gl 498DS having a nick or a gap of one to six nucleotides that begins at any one of positions 8 to 12 of the sense strand.
  • Figure 8 shows knockdown activity of a LacZ RISC dsRNA having a nick or a gap of one to six nucleotides that begins at any one of positions 8 to 14 of the sense strand.
  • Figure 11 shows the percent knockdown in influenza viral titers using influenza specific mdRNA against influenza strain WSN.
  • Figure 12 shows the in vivo reduction in PR8 influenza viral titers using influenza specific mdRNA as measured by TCID 50 .
  • dsRNA nicked or gapped double-stranded RNA
  • RISC RNA interference pathway
  • partially duplexed dsRNA molecules described herein are capable of initiating an RNA interference cascade that modifies ⁇ e.g., reduces) expression of a target messenger RNA (mRNA), such as a human vascular endothelial growth factor receptor (STAT3) mRNA.
  • mRNA target messenger RNA
  • STAT3 human vascular endothelial growth factor receptor
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid molecule to proceed, for example, RNAi activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid molecule (e.g., dsRNA) 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, or under conditions in which the assays are performed in the case of in vitro assays (e.g., hybridization assays).
  • nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable or to specifically bind. That is, two or more nucleic acid molecules may be less than fully complementary and is indicated by a percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule.
  • Perfectly or “fully” complementary nucleic acid molecules means those in which a certain number of nucleotides of a first nucleic acid molecule hydrogen bond (anneal) with the same number of residues in a second nucleic acid molecule to form a contiguous double-stranded region.
  • two or more fully complementary nucleic acid molecule strands can have the same number of nucleotides (i.e., have the same length and form one double-stranded region, with or without an overhang) or have a different number of nucleotides (e.g. , one strand may be shorter than but fully contained within another strand or one strand may overhang the other strand).
  • RNA refers to a nucleic acid molecule comprising at least one ribonucleotide molecule.
  • ribonucleotide refers to a nucleotide with a hydroxyl group at the 2'-position of a ⁇ -D-ribofuranose moiety.
  • RNA includes double-stranded (ds) RNA, single-stranded (ss) RNA, isolated RNA (such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), altered RNA (which differs from naturally occurring RNA by the addition, deletion, substitution or alteration of one or more nucleotides), or any combination thereof.
  • such altered RNA can include addition of non-nucleotide material, such as at one or both ends of an RNA molecule, internally at one or more nucleotides of the RNA, or any combination thereof.
  • Nucleotides in RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as naturally occurring nucleotides, non-naturally occurring nucleotides, chemically-modified nucleotides, deoxynucleotides, or any combination thereof.
  • RNAs may be referred to as analogs or analogs of RNA containing standard nucleotides (i.e., standard nucleotides, as used herein, are considered to be adenine, cytidine, guanidine, thymidine, and uridine).
  • standard nucleotides i.e., standard nucleotides, as used herein, are considered to be adenine, cytidine, guanidine, thymidine, and uridine).
  • dsRNA molecules in addition to at least one ribonucleotide, can further include substitutions, chemically-modified nucleotides, and non-nucleotides. In certain embodiments, dsRNA molecules comprise ribonucleotides up to about 100% of the nucleotide positions.
  • dsRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example, meroduplex RNA (mdRNA), nicked dsRNA (ndsRNA), gapped dsRNA (gdsRNA), short interfering nucleic acid (siNA), siRNA, micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering substituted oligonucleotide, short interfering modified oligonucleotide, chemically-modified dsRNA, post-transcriptional gene silencing RNA (ptgsRNA), or the like.
  • mdRNA meroduplex RNA
  • ndsRNA nicked dsRNA
  • gdsRNA gapped dsRNA
  • siNA short interfering nucleic acid
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • ptgsRNA post-
  • large dsRNA refers to any double-stranded RNA longer than about 40 base pairs (bp) to about 100 bp or more, particularly up to about 300 bp to about 500 bp.
  • the sequence of a large dsRNA may represent a segment of an mRNA or an entire mRNA.
  • a double-stranded structure may be formed by a self-complementary nucleic acid molecule or by annealing of two or more distinct complementary nucleic acid molecule strands.
  • a dsRNA comprises two separate oligonucleotides, comprising a first strand (antisense) and a second strand (sense), wherein the antisense and sense strands are self- complementary (i.e., each strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the other strand and the two separate strands form a duplex or double-stranded structure, for example, wherein the double-stranded region is about 15 to about 24 base pairs or about 26 to about 40 base pairs); the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g.
  • a human STAT3 mRNA of SEQ ID NO:1158, 1159, or 1160 comprises a nucleotide sequence corresponding (i.e., homologous) to the target nucleic acid sequence or a portion thereof (e.g., a sense strand of about 15 to about
  • nucleic acid 25 nucleotides or about 26 to about 40 nucleotides corresponds to the target nucleic acid or a portion thereof).
  • the dsRNA is assembled from a single oligonucleotide in which the self-complementary sense and antisense strands of the dsRNA are linked together by a nucleic acid based-linker or a non-nucleic acid-based linker.
  • the first (antisense) and second (sense) strands of the dsRNA molecule are covalently linked by a nucleotide or non-nucleotide linker as described herein and known in the art.
  • a first dsRNA molecule is covalently linked to at least one second dsRNA molecule by a nucleotide or non-nucleotide linker known in the art, wherein the first dsRNA molecule can be linked to a plurality of other dsRNA molecules that can be the same or different, or any combination thereof.
  • the linked dsRNA may include a third strand that forms a meroduplex with the linked dsRNA.
  • dsRNA molecules described herein form a meroduplex RNA (mdRNA) having three or more strands such as, for example, an A' (first or antisense) strand, 'Sl' (second) strand, and 'S2' (third) strand in which the 'Sl' and 'S2' strands are complementary to and form base pairs (bp) with non-overlapping regions of the A' strand (e.g., an mdRNA can have the form of A:S1S2).
  • mdRNA meroduplex RNA
  • the double-stranded region formed by the annealing of the 'Sl' and A' strands is distinct from and non-overlapping with the double-stranded region formed by the annealing of the 'S2' and A' strands.
  • An mdRNA molecule is a "gapped" molecule, i.e., it contains a "gap” ranging from 0 nucleotides up to about 10 nucleotides (or a gap of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides).
  • the A: Sl duplex is separated from the A:S2 duplex by a gap of zero nucleotides (i.e., a nick in which only a phosphodiester bond between two nucleotides is broken or missing in the polynucleotide molecule) between the A: Sl duplex and the A:S2 duplex - which can also be referred to as nicked dsRNA (ndsRNA).
  • a gap of zero nucleotides i.e., a nick in which only a phosphodiester bond between two nucleotides is broken or missing in the polynucleotide molecule
  • A:S 1 S2 may be comprised of a dsRNA having at least two double-stranded regions that combined total about 14 base pairs to about 40 base pairs and the double-stranded regions are separated by a gap of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides, optionally having blunt ends, or
  • A:S1S2 may comprise a dsRNA having at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands wherein at least one of the double-stranded regions optionally has from 5 base pairs to 13 base pairs.
  • a dsRNA or large dsRNA may include a substitution or modification in which the substitution or modification may be in a phosphate backbone bond, a sugar, a base, or a nucleoside.
  • nucleoside substitutions can include natural non-standard nucleosides (e.g., 5-methyluridine or 5-methylcytidine or a 2-thioribothymidine), and such backbone, sugar, or nucleoside modifications can include an alkyl or heteroatom substitution or addition, such as a methyl, alkoxyalkyl, halogen, nitrogen or sulfur, or other modifications known in the art.
  • RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
  • dsRNA molecules of this disclosure can be used to epigenetically silence genes at the post-transcriptional level or the pre -transcriptional level or any combination thereof.
  • target nucleic acid refers to any nucleic acid sequence whose expression or activity is to be altered (e.g., STAT3).
  • the target nucleic acid can be DNA, RNA, or analogs thereof, and includes single, double, and multi-stranded forms.
  • target site or “target sequence” is meant a sequence within a target nucleic acid (e.g., mRNA) that, when present in an RNA molecule, is “targeted” for cleavage by RNAi and mediated by a dsRNA construct of this disclosure containing a sequence within the antisense strand that is complementary to the target site or sequence.
  • sense region or “sense strand” is meant one ore more nucleotide sequences of a dsRNA molecule having complementarity to one or more antisense regions of the dsRNA molecule.
  • the sense region of a dsRNA molecule comprises a nucleic acid sequence having homology or identity to a target sequence, such as STAT3.
  • antisense region or “antisense strand” is meant a nucleotide sequence of a dsRNA molecule having complementarity to a target nucleic acid sequence, such as STAT3.
  • the antisense region of a dsRNA molecule can comprise nucleic acid sequence region having complementarity to one or more sense strands of the dsRNA molecule.
  • Analog refers to a compound that is structurally similar to a parent compound (e.g., a nucleic acid molecule), but differs slightly in composition (e.g., one atom or functional group is different, added, or removed).
  • the analog may or may not have different chemical or physical properties than the original compound and may or may not have improved biological or chemical activity.
  • the analog may be more hydrophilic or it may have altered activity as compared to a parent compound.
  • the analog may mimic the chemical or biological activity of the parent compound (i.e., it may have similar or identical activity), or, in some cases, may have increased or decreased activity.
  • the analog may be a naturally or non- naturally occurring (e.g., chemically-modified or recombinant) variant of the original compound.
  • An example of an RNA analog is an RNA molecule having a non-standard nucleotide, such as 5-methyuridine or 5-methylcytidine or 2-thioribothymidine, which may impart certain desirable properties (e.g., improve stability, bioavailability, minimize off-target effects or interferon response).
  • universal base refers to nucleotide base analogs that form base pairs with each of the standard DNA/RNA bases with little discrimination between them. A universal base is thus interchangeable with all of the standard bases when substituted into a nucleotide duplex (see, e.g., Loakes et ah, J. MoI. Bio. 270:426, 1997).
  • Examplary universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, or nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucleic Acids Res. 29:2431, 2001).
  • the extent of silencing may be determined by methods described herein and known in the art (see, e.g., PCT Publication No. WO 99/32619; Elbashir et al, EMBO J. 20:6X77, 2001).
  • quantification of gene expression permits detection of various amounts of inhibition that may be desired in certain embodiments of this disclosure, including prophylactic and therapeutic methods, which will be capable of knocking down target gene expression, in terms of mRNA level or protein level or activity, for example, by equal to or greater than 10%, 30%, 50%, 75% 90%, 95% or 99% of baseline (i.e., normal) or other control levels, including elevated expression levels as may be associated with particular disease states or other conditions targeted for therapy.
  • a therapeutically effective amount of a therapeutic compound can decrease, for example, atheromatous plaque size or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such therapeutically effective amounts based on such factors as the subject's size, the severity of symptoms, and the particular composition or route of administration selected.
  • the nucleic acid molecules of the instant disclosure individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein.
  • the dsRNA molecules can be administered to a patient or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs, under conditions suitable for treatment.
  • cycloalkyl refers to a saturated cyclic hydrocarbon ring system containing from 3 to 12 carbon atoms that may be optionally substituted. Exemplary embodiments include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. In certain embodiments, the cycloalkyl group is cyclopropyl. In another embodiment, the (cycloalkyl)alkyl groups contain from 3 to 12 carbon atoms in the cyclic portion and 1 to 6 carbon atoms in the alkyl portion. In certain embodiments, the (cycloalkyl)alkyl group is cyclopropylmethyl. The alkyl groups are optionally substituted with from one to three substituents selected from the group consisting of halogen, hydroxy and amino.
  • alkenyl refers to an unsaturated branched, straight-chain or cyclic alkyl group having 2 to 15 carbon atoms and having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond(s).
  • Certain embodiments include ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2- propenyl, 1-pentenyl, 2-pentenyl, 4-pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 1- heptenyl, 2-heptenyl, 1-octenyl, 2-octenyl, 1,3-octadienyl, 2-nonenyl, 1,3-nonadienyl, 2-decenyl, etc., or the like.
  • the alkenyl group may be substituted or unsubstituted.
  • alkynyl refers to an unsaturated branched, straight-chain, or cyclic alkyl group having 2 to 10 carbon atoms and having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne.
  • exemplary alkynyls include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3- butynyl, 1-pentynyl, 2-pentynyl, 4-pentynyl, 1-octynyl, 6-methyl-l-heptynyl, 2-decynyl, or the like.
  • the alkynyl group may be substituted or unsubstituted.
  • hydroxyalkyl alone or in combination, refers to an alkyl group as previously defined, wherein one or several hydrogen atoms, preferably one hydrogen atom has been replaced by a hydroxyl group. Examples include hydroxymethyl, hydroxy ethyl and 2- hydroxy ethyl.
  • dialkylaminoalkyl refers to alkylamino groups attached to an alkyl group. Examples include, but are not limited to, N,N-dimethylaminomethyl, N,N-dimethylaminoethyl N,N-dimethylaminopropyl, and the like.
  • dialkylaminoalkyl also includes groups where the bridging alkyl moiety is optionally substituted.
  • haloalkyl refers to an alkyl group substituted with one or more halo groups, for example chloromethyl, 2-bromoethyl, 3-iodopropyl, trifluoromethyl, perfluoropropyl, 8- chlorononyl, or the like.
  • alkyl refers to a saturated straight- or branched-chain hydrocarbyl radical of 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, 2-methylpentyl, n-hexyl, and so forth.
  • Alkylene is the same as alkyl except that the group is divalent.
  • alkoxy includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom.
  • the alkoxy group contains 1 to about 10 carbon atoms.
  • Embodiments of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups.
  • Embodiments of substituted alkoxy groups include halogenated alkoxy groups.
  • the alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
  • alkoxyalkyl refers to an alkylene group substituted with an alkoxy group.
  • methoxyethyl CH 3 OCH 2 CH 2 -
  • ethoxymethyl CH 3 CH 2 OCH 2 -
  • aryl refers to monocyclic or bicyclic aromatic hydrocarbon groups having from 6 to 12 carbon atoms in the ring portion, for example, phenyl, naphthyl, biphenyl and diphenyl groups, each of which may be substituted with, for example, one to four substituents such as alkyl; substituted alkyl as defined above, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyloxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, nitro, cyano, carboxy, carboxyalkyl, carbamyl, carbamoyl and aryloxy.
  • Specific embodiments of aryl groups in accordance with the present disclosure include phenyl, substituted phenyl, naphthyl, biphenyl, and diphenyl.
  • aroyl refers to an aryl radical derived from an aromatic carboxylic acid, such as optionally substituted benzoic or naphthoic acids.
  • aralkyl refers to an aryl group bonded to the 2-pyridinyl ring or the 4-pyridinyl ring through an alkyl group, preferably one containing 1 to 10 carbon atoms.
  • a preferred aralkyl group is benzyl.
  • trifluoromethyl refers to -CF 3 .
  • trifluoromethoxy refers to -OCF 3 .
  • hydroxyl refers to -OH or -0 " .
  • nitrile or “cyano” as used herein refers to the group -CN.
  • nitro as used herein alone or in combination refers to a -NO 2 group.
  • amino refers to the group -NR 9 R 9 , wherein R 9 may independently be hydrogen, alkyl, aryl, alkoxy, or heteroaryl.
  • aminoalkyl as used herein represents a more detailed selection as compared to “amino” and refers to the group -NR'R', wherein R' may independently be hydrogen or (C 1 -C 4 ) alkyl.
  • dialkylamino refers to an amino group having two attached alkyl groups that can be the same or different.
  • carbonylamino refers to the group -NRZ-CO-CH 2 -R', wherein R' may be independently selected from hydrogen or (C 1 -C 4 ) alkyl.
  • carbamoyl refers to -0-C(O)NH 2 .
  • Exemplary monocyclic heterocyclo groups include pyrrolidinyl, pyrrolyl, indolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, tetrahydrofuryl, thienyl, piperidinyl, piperazinyl, azepinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, dioxanyl, triazinyl and triazolyl.
  • Preferred bicyclic heterocyclo groups include benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, benzimidazolyl, benzofuryl, indazolyl, benzisothiazolyl, isoindolinyl and tetrahydroquinolinyl.
  • heterocyclo groups may include indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl and pyrimidyl.
  • Substituted refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s).
  • STAT3 Signal transducer and activator of transcription 3
  • dsRNA Molecules Exemplary dsRNA Molecules
  • STAT3 mRNA or RNA sequences or sense strands means an STAT3 RNA isoform as set forth in SEQ ID NO: 1158, 11559, or 1160, 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 NOS:1158, 1159, or 1160.
  • the comparison of sequences and determination of percent identity between two or more sequences can be accomplished using a mathematical algorithm, such as BLAST and Gapped BLAST programs at their default parameters (e.g., BLASTN, see www.ncbi.nlm.nih.gov/BLAST; see also Altschul et al, J. MoL Biol. 275:403-410, 1990).
  • the instant disclosure provides an mdRNA molecule, comprising a first strand that is complementary to a STAT3 mRNA as set forth in SEQ ID NO: 1158, 1159, or 1160, and a second strand and a third strand that are each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein (a) the mdRNA molecule optionally includes at least one double-stranded region comprises from about 5 base pairs to 13 base pairs, or (b) wherein the combined double-stranded regions total about 5 base pairs to about 40 base pairs and the mdRNA molecule optionally has blunt ends; wherein at least one pyrimidine of the mdRNA is a pyrimidine nucleoside according to Formula I or II:
  • At least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -OH, or R 1 is methyl, R 2 is -OH, and R 8 is S. In certain embodiments, at least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is - O-methyl, or R 1 is methyl, R 2 is -O-methyl, and R 8 is O. In other embodiments, the internucleoside linking group covalently links from about 5 to about 40 nucleosides.
  • the instant disclosure provides an mdRNA molecule, comprising a first strand that is complementary to a STAT3 mRNA as set forth in SEQ ID NO: 1158, 1159, or 1160, and a second strand and a third strand that are each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein the mdRNA molecule optionally includes at least one double-stranded region comprises from 5 base pairs to 13 base pairs.
  • the instant disclosure provides an mdRNA molecule having a first strand that is complementary to a STAT3 mRNA as set forth in SEQ ID NO: 1158, 1159, or 1160, and a second strand and a third strand that are each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein the combined double- stranded regions total about 15 base pairs to about 40 base pairs and the mdRNA molecule optionally has blunt ends.
  • the gap comprises at least one unpaired nucleotide in the first strand positioned between the double-stranded regions formed by the second and third strands when annealed to the first strand, or the gap is a nick.
  • the nick or gap is located 10 nucleotides from the 5 '-end of the first (antisense) strand or at the Argonaute cleavage site.
  • the meroduplex nick or gap is positioned such that the thermal stability is maximized for the first and second strand duplex and for the first and third strand duplex as compared to the thermal stability of such meroduplexes having a nick or gap in a different position.
  • any of the aspects or embodiments disclosed herein would be useful in treating STAT3-associated diseases or disorders, such as one or more hyperproliferative disorders, for example leukemia, lymphoma, breast cancer, prostate cancer, multiple myeloma, and head and neck cancer, as well as tumor proliferation.
  • STAT3-associated diseases or disorders such as one or more hyperproliferative disorders, for example leukemia, lymphoma, breast cancer, prostate cancer, multiple myeloma, and head and neck cancer, as well as tumor proliferation.
  • the dsRNA comprises at least three strands in which the first strand comprises about 5 nucleotides to about 40 nucleotides, and the second and third strands include each, individually, about 5 nucleotides to about 20 nucleotides, wherein the combined length of the second and third strands is about 15 nucleotides to about 40 nucleotides.
  • the dsRNA comprises at least two strands in which the first strand comprises about 15 nucleotides to about 24 nucleotides or about 25 nucleotides to about 40 nucleotides.
  • the first strand comprises about 15 to about 24 nucleotides or about 25 nucleotides to about 40 nucleotides and is complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides of human STAT3 mRNA as set forth in SEQ ID NO: 1158, 1159, or 1160.
  • the first strand comprises about 15 to about 24 nucleotides or about 25 nucleotides to about 40 nucleotides and is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence that is complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides of human STAT3 mRNA as set forth in SEQ ID NO:1158- 1160.
  • the first strand will be complementary to a second strand or a second and third strand or to a plurality of strands.
  • the first strand and its complements will be able to form dsRNA and mdRNA molecules of this disclosure, but only about 19 to about 25 nucleotides of the first strand comprise a sequence complementary to a STAT3 mRNA.
  • a Dicer substrate dsRNA can have about 25 nucleotides to about 40 nucleotides, but with only 19 nucleotides of the antisense (first) strand being complementary to a STAT3 mRNA.
  • the first strand having complementarity to a STAT3 mRNA in about 19 nucleotides to about 25 nucleotides will have one, two, or three mismatches with a STAT3 mRNA, such as a sequence set forth in SEQ ID NO: 1158, 1159, or 1160, or the first strand of 19 nucleotides to about 25 nucleotides, that for example activates or is capable of loading into RISC, will have at least 80% identity with the corresponding nucleotides found in a STAT3 mRNA, such as the sequence set forth in SEQ ID NO: 1158, 1159, or 1160.
  • dsRNA molecules which can be used to design mdRNA molecules and can optionally include substitutions or modifications as described herein are provided in the Sequence Listings attached herewith, which is herein incorporated by reference (text file "07-R064PCT_Sequence_Listing," created February 15, 2008 and having a size of
  • dsRNA molecules of this disclosure provide a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules ⁇ i.e., having standard nucleotides) that are exogenously delivered.
  • the use of dsRNA molecules of this disclosure can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect ⁇ e.g., reducing or silencing STAT3 expression) since dsRNA molecules of this disclosure tend to have a longer half-life in serum.
  • certain substitutions and modifications can improve the bioavailability of dsRNA by targeting particular cells or tissues or improving cellular uptake of the dsRNA molecules.
  • substituted and modified dsRNA can also minimize the possibility of activating the interferon response in, for example, humans.
  • a dsRNA molecule of this disclosure has at least one uridine, at least three uridines, or each and every uridine ⁇ i.e., all uridines) of the first (antisense) strand of that is a 5-methyluridine, 2-thioribothymidine, 2'-O-methyl-5-methyluridine, or any combination thereof.
  • the dsRNA molecule or analog thereof of this disclosure has at least one uridine, at least three uridines, or each and every uridine of the second (sense) strand of the dsRNA is a 5-methyluridine, 2-thioribothymidine, 2'-O-methyl-5-methyluridine, or any combination thereof.
  • the dsRNA molecule of this disclosure has at least one uridine, at least three uridines, or each and every uridine of the third (sense) strand of the dsRNA is a 5-methyluridine, 2-thioribothymidine, 2'-O-methyl-5-methyluridine, or any combination thereof.
  • the dsRNA molecule of this disclosure has at least one uridine, at least three uridines, or each and every uridine of both the first (antisense) and second (sense) strands; of both the first (antisense) and third (sense) strands; of both the second (sense) and third (sense) strands; or all of the first (antisense), second (sense) and third (sense) strands of the dsRNA are a 5-methyluridine, 2-thioribothymidine, 2'-O-methyl-5- methyluridine, or any combination thereof.
  • the double-stranded region of a dsRNA molecule has at least three 5-methyluridines, 2-thioribothymidine, 2'-O-methyl-5- methyluridine, or any combination thereof.
  • dsRNA molecules comprise ribonucleotides at about 5% to about 95% of the nucleotide positions in one strand, both strands, or any combination thereof.
  • a dsRNA molecule that decreases expression of a STAT3 gene by RNAi according to the instant disclosure further comprises one or more natural or synthetic non-standard nucleoside.
  • the dsRNA has at least one double-stranded region ranging in length from about 26 to about 40 base pairs or about 27 to about 30 base pairs or about 30 to about 35 base pairs.
  • the dsRNA molecule or analog thereof has an overhang of one to four nucleotides on one or both 3'-ends, such as an overhang comprising a deoxyribonucleotide or two deoxyribonucleotides (e.g., thymidine).
  • dsRNA molecule or analog thereof has a blunt end at one or both ends of the dsRNA.
  • the 5'-end of the first or second strand is phosphorylated.
  • At least one of the at least two pyrimidine nucleosides in which R 2 is not -H or -OH is located at a 3 '-end or a 5 '-end within the double-stranded region of at least one strand of the dsRNA molecule, and wherein at least one of the at least two pyrimidine nucleosides in which R 2 is not -H or -OH is located internally within a strand of the dsRNA molecule.
  • At least one of the at least two pyrimidine nucleosides in which R 2 is not -H or -OH is located at a 3 '-end or a 5 '-end of at least one strand of the dsRNA molecule, and wherein at least one of the at least two pyrimidine nucleosides in which R 2 is not -H or -OH is located internally within a strand of the dsRNA molecule.
  • a dsRNA molecule or analog thereof of Formula I or II according to the instant disclosure further comprises a terminal cap substituent on one or both ends of the first strand or second strand, such as an alkyl, abasic, deoxy abasic, glyceryl, dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety, or any combination thereof.
  • a terminal cap substituent on one or both ends of the first strand or second strand such as an alkyl, abasic, deoxy abasic, glyceryl, dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety, or any combination thereof.
  • one or more internucleoside linkage can be optionally modified.
  • the dsRNAs comprise at least two or more substituted pyrimidine nucleosides can each be independently selected wherein R 1 comprises any chemical modification or substitution as contemplated herein, for example an alkyl (e.g., methyl), halogen, hydroxy, alkoxy, nitro, amino, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, carboxyalkyl, alkoxyalkyl, carboxy, carbonyl, alkanoylamino, carbamoyl, carbonylamino, alkylsulfonylamino, or heterocyclo group.
  • R 1 comprises any chemical modification or substitution
  • the linker portion may have a clover- leaf tRNA structure. Even if the linker has a length that would hinder pairing of the stem portion, it is possible, for example, to construct the linker portion to include introns so that the introns are excised during processing of a precursor RNA into mature RNA, thereby allowing pairing of the stem portion.
  • either end (head or tail) of RNA with no loop structure may have a low molecular weight RNA.
  • these low molecular weight RNAs may include a natural RNA molecule, such as tRNA, rRNA or viral RNA, or an artificial RNA molecule.
  • dsRNAs of this disclosure can comprise one or more sense (second) strands that are homologous or correspond to a sequence of a target gene (e.g., a
  • a dsRNA will have from one to all uridines substituted with ribothymidine and have up to about 75% 2'-methoxy substitutions (and not at the Argonaute cleavage site). In still other embodiments, a dsRNA will have from one to all uridines substituted with ribothymidine and have up to about 100% 2'-fluoro substitutions. In further embodiments, a dsRNA will have from one to all uridines substituted with ribothymidine and have up to about 75% 2'-deoxy substitutions. In further embodiments, a dsRNA will have up to about 75% LNA substitutions and have up to about 75% 2'-methoxy substitutions.
  • a dsRNA will have from one to all uridines substituted with ribothymidine, up to about 75% LNA substitutions, up to about 75% 2'-methoxy, up to about 100% 2'-fluoro, and up to about 75% 2'-deoxy substitutions. In other embodiments, a dsRNA will have up to about 75% LNA substitutions, up to about 75% 2'-methoxy substitutions, and up to about 100% 2'-fluoro substitutions. In further embodiments, a dsRNA will have up to about 75% LNA substitutions, up to about 75% 2'-methoxy substitutions, and up to about 75% 2'-deoxy substitutions.
  • any of these multiple modification embodiments may have these multiple modifications on one strand, two strands, three strands, a plurality of strands, or all strands.
  • the dsRNA must have gene silencing activity.
  • dsRNA disclosed herein can include between about 1 universal-binding nucleotide and about 10 universal-binding nucleotides.
  • the presently disclosed dsRNA may comprise a sense strand that is homologous to a sequence of a STAT3 gene and an antisense strand that is complementary to the sense strand, with the proviso that at least one nucleotide of the antisense or sense strand of the otherwise complementary dsRNA duplex has one or more universal-binding nucleotide.
  • Exemplary molecules of the instant disclosure are recombinantly produced, chemically synthesized, or a combination thereof.
  • Oligonucleotides e.g. , certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides
  • Oligonucleotides are synthesized using protocols known in the art, for example as described in Caruthers et al, Methods in Enzymol. 211:3-19, 1992;
  • RNA including certain dsRNA molecules and analogs thereof of this disclosure, can be made using the procedure as described in Usman et al, J. Am. Chem. Soc. 109:7845, 1987; Scaringe et al,
  • nucleic acid molecules of the present disclosure can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al, Science 256:9923, 1992; Draper et al., PCT Publication No. WO 93/23569; Shabarova et al., Nucleic Acids Res. 19:4247, 1991; Bellon et al., Nucleosides & Nucleotides 16:951, 1997; Bellon et al., Bioconjugate Chem. 5:204, 1997), or by hybridization following synthesis or deprotection.
  • mismatch portions contained in the double-stranded region of dsRNAs may include from about 1 to 7, or about 1 to 5 mismatches.
  • the double- stranded region of dsRNAs of this disclosure may contain both bulge and mismatched portions in the approximate numerical ranges specified herein.
  • a dsRNA or analog thereof of this disclosure may be further comprised of a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the dsRNA to the antisense region of the dsRNA.
  • a nucleotide linker can be a linker of more than about 2 nucleotides length up to about 10 nucleotides in length.
  • the nucleotide linker can be a nucleic acid aptamer.
  • aptamer or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting.
  • an aptamer can be a nucleic acid molecule that binds to a target molecule wherein the target molecule does not naturally bind to a nucleic acid.
  • the target molecule can be any molecule of interest.
  • the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein.
  • a non-nucleotide linker may be comprised of an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units).
  • polyethylene glycols such as those having between 2 and 100 ethylene glycol units.
  • Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 18:6353, 1990, and Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J. Am. Chem. Soc. 113:6324, 1991; Richardson and Schepartz, J. Am. Chem. Soc. 113:5109, 1991; Ma et al,
  • the synthesis of a dsRNA molecule of this disclosure comprises: (a) synthesis of a first (antisense) strand and synthesis of a second (sense) strand and a third (sense) strand that are each complementary to non-overlapping regions of the first strand; and (b) annealing the first, second and third strands together under conditions suitable to obtain a dsRNA molecule.
  • synthesis of the first, second and thirdstrands of a dsRNA molecule is by solid phase oligonucleotide synthesis.
  • synthesis of the first, second ,and third strands of a dsRNA molecule is by solid phase tandem oligonucleotide synthesis.
  • oligonucleotides can be modified at the sugar moiety to enhance stability or prolong biological activity by increasing nuclease resistance.
  • Representative sugar modifications include 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O- methyl, 2'-O-allyl, or 2'-H.
  • Other modifications to enhance stability or prolong biological activity can be internucleoside linkages, such as phosphorothioate, or base-modifications, such as locked nucleic acids (see, e.g., U.S. Patent Nos.
  • dsRNA molecules of the instant disclosure can be modified to increase nuclease resistance or duplex stability while substantially retaining or having enhanced RNAi activity as compared to unmodified dsRNA.
  • conjugate molecules contemplated by the instant disclosure that can be attached to a dsRNA or analog thereof of this disclosure are described in Vargeese et al, U.S. Patent Application Publication No. 2003/0130186, and U.S. Patent Application Publication No. 2004/0110296.
  • a conjugate molecule is covalently attached to a dsRNA or analog thereof that decreases expression of a STAT3 gene by RNAi via a biodegradable linker.
  • a conjugate molecule can be attached at the 3 '-end of either the sense strand, the antisense strand, or both strands of a dsRNA molecule provided herein.
  • a conjugate molecule can be attached at the 5 '-end of either the sense strand, the antisense strand, or both strands of the dsRNA or analog thereof. In yet another embodiment, a conjugate molecule is attached at both the 3 '-end and 5 '-end of either the sense strand, the antisense strand, or both strands of a dsRNA molecule, or any combination thereof. In further embodiments, a conjugate molecule of this disclosure comprises a molecule that facilitates delivery of a dsRNA or analog thereof into a biological system, such as a cell.
  • dsRNA of this disclosure having various conjugates to determine whether the dsRNA-conjugate possesses improved properties (e.g., pharmacokinetic profiles, bioavailability, stability) while maintaining the ability to mediate RNAi in, for example, an animal model as described herein or generally known in the art.
  • improved properties e.g., pharmacokinetic profiles, bioavailability, stability
  • nucleotide sequence of every possible gene variant (including mRNA splice variants) targeted by the dsRNA or analog thereof is selected from a conserved region or consensus sequence of a STAT3 gene.
  • nucleotide sequence of the dsRNA may be selectively or preferentially targeted to a certain sequence contained in an mRNA splice variant of a STAT3 gene.
  • a dsRNA as provided herein will exhibit a greater stability, minimal interferon response, and little or no "off-target” binding.
  • Still further embodiments provide methods for selecting more efficacious dsRNA by using one or more reporter gene constructs comprising a constitutive promoter, such as a cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter, operably fused to, and capable of altering the expression of one or more reporter genes, such as a luciferase, chloramphenicol (CAT), or ⁇ -galactosidase, which, in turn, is operably fused in-frame with a dsRNA (such as one having a length between about 15 base-pairs and about 40 base-pairs or from about 5 nucleotides to about 24 nucleotides, or about 25 nucleotides to about 40 nu
  • a constitutive promoter such as a cytomegalovirus (CMV) or phosphog
  • Individual reporter gene expression constructs may be co-transfected with one or more dsRNA or analog thereof.
  • STAT3 may be determined by comparing the measured reporter gene activity in cells transfected with or without a dsRNA molecule of interest.
  • Certain embodiments disclosed herein provide methods for selecting one or more modified dsRNA molecule(s) that employ the step of predicting the stability of a dsRNA duplex.
  • a prediction is achieved by employing a theoretical melting curve wherein a higher theoretical melting curve indicates an increase in dsRNA duplex stability and a concomitant decrease in cytotoxic effects.
  • stability of a dsRNA duplex may be determined empirically by measuring the hybridization of a single RNA analog strand as described herein to a complementary target gene within, for example, a polynucleotide array. The melting temperature (i.e., the T m value) for each modified RNA and complementary RNA immobilized on the array can be determined and, from this T m value, the relative stability of the modified RNA pairing with a complementary RNA molecule determined.
  • nucleotide substitutions e.g., 5-methyluridine for uridine
  • further modifications e.g., a ribose modification at the 2'-position
  • one or more anti-codon within an antisense strand of a dsRNA molecule or analog thereof is substituted with a universal-binding nucleotide in a second or third position in the anti-codon of the antisense strand.
  • a universal-binding nucleotide for a first or second position, the one or more first or second position nucleotide-pair substitution allows the substituted dsRNA molecule to specifically bind to mRNA wherein a first or a second position nucleotide-pair substitution has occurred, wherein the one or more nucleotide-pair substitution results in an amino acid change in the corresponding gene product.
  • any of these methods of identifying dsRNA of interest can also be used to examine a dsRNA that decreases expression of a STAT3 gene by RNA interference, comprising a first strand that is complementary to a STAT3 mRNA as set forth in SEQ ID NO: 1158, 1159, or 1160, and a second and third strand that have non-overlapping complementarity to the first strand, wherein the first and at least one of the second or third strand form a double-stranded region of about 5 to about 13 base pairs; wherein at least one pyrimidine of the dsRNA comprises a pyrimidine nucleoside according to Formula I or II:
  • R 1 and R 2 are each independently a -H, -OH, -OCH 3 , -OCH 2 OCH 2 CH 3 ,
  • dsRNA of the instant disclosure are designed to target a STAT3 gene (including one or more mRNA splice variant thereof) that is expressed at an elevated level or continues to be expressed when it should not, and is a causal or contributing factor associated with one or more hyperproliferative disorders, for example leukemia, lymphoma, breast cancer, prostate cancer, multiple myeloma, and head and neck cancer, as well as tumor proliferation.
  • a dsRNA or analog thereof of this disclosure will effectively downregulate expression of a STAT3 gene to levels that prevent, alleviate, or reduce the severity or recurrence of one or more associated disease symptoms.
  • dsRNAs of this disclosure may be targeted to lower expression of STAT3, which can result in upregulation of a "downstream" gene whose expression is negatively regulated, directly or indirectly, by a STAT3 protein.
  • the dsRNA molecules of the instant disclosure comprise useful reagents and can be used in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.
  • aqueous suspensions contain dsRNA of this disclosure in admixture with suitable excipients, such as suspending agents or dispersing or wetting agents.
  • suitable excipients such as suspending agents or dispersing or wetting agents.
  • suspending agents include sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.
  • Representative dispersing or wetting agents include naturally-occurring phosphatides (e.g., lecithin), condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate).
  • naturally-occurring phosphatides e.g., lecithin
  • condensation products of an alkylene oxide with fatty acids e.g., polyoxyethylene stearate
  • condensation products of ethylene oxide with long chain aliphatic alcohols e.g., heptadecaethyleneoxycetan
  • the aqueous suspensions can optionally contain one or more preservatives (e.g., ethyl or n-propyl-p- hydroxybenzoate), one or more coloring agents, one or more flavoring agents, or one or more sweetening agents (e.g., sucrose, saccharin).
  • preservatives e.g., ethyl or n-propyl-p- hydroxybenzoate
  • coloring agents e.g., one or more coloring agents
  • one or more flavoring agents e.g., sucrose, saccharin
  • sweetening agents e.g., sucrose, saccharin
  • dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide dsRNA of this disclosure in admixture with a dispersing or wetting agent, suspending agent and optionally one or more preservative, coloring agent, flavoring agent, or sweetening agent.
  • compositions prepared for storage or administration that include a pharmaceutically effective amount of a desired compound in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., A.R. Gennaro edit., 1985, hereby incorporated by reference herein.
  • pharmaceutical compositions of this disclosure can optionally include preservatives, antioxidants, stabilizers, dyes, flavoring agents, or any combination thereof.
  • Exemplary preservatives include sodium benzoate, sorbic acid, chlorobutanol, and esters of p-hydroxybenzoic acid.
  • compositions of the instant disclosure can be effectively employed as pharmaceutically-acceptable formulations.
  • Pharmaceutically-acceptable formulations prevent, alter the occurrence or severity of, or treat (alleviate one or more symptom(s) to a detectable or measurable extent) of a disease state or other adverse condition in a subject.
  • a pharmaceutically acceptable formulation includes salts of the above compounds, e.g., acid addition salts, such as salts of hydrochloric acid, hydrobromic acid, acetic acid, or benzene sulfonic acid.
  • a pharmaceutical composition or formulation refers to a composition or formulation in a form suitable for administration into a cell, or a subject such as a human (e.g., systemic administration).
  • compositions of the present disclosure having an amount of dsRNA sufficient to treat or prevent a disorder associated with STAT3 gene expression are, for example, suitable for topical (e.g., creams, ointments, skin patches, eye drops, ear drops) application or administration.
  • topical e.g., creams, ointments, skin patches, eye drops, ear drops
  • Other routes of administration include oral, parenteral, sublingual, bladder wash-out, vaginal, rectal, enteric, suppository, nasal, and inhalation.
  • parenteral includes subcutaneous, intravenous, intramuscular, intraarterial, intraabdominal, intraperitoneal, intraarticular, intraocular or retrobulbar, intraaural, intrathecal, intracavitary, intracelial, intraspinal, intrapulmonary or transpulmonary, intrasynovial, and intraurethral injection or infusion techniques.
  • the pharmaceutical compositions of the present disclosure are formulated to allow the dsRNA contained therein to be bioavailable upon administration to a subject.
  • dsRNA of this disclosure can be formulated as oily suspensions or emulsions (e.g., oil-in-water) by suspending dsRNA in, for example, a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil) or a mineral oil (e.g., liquid paraffin).
  • a vegetable oil e.g., arachis oil, olive oil, sesame oil or coconut oil
  • a mineral oil e.g., liquid paraffin
  • Suitable emulsifying agents can be naturally-occurring gums (e.g.
  • gum acacia or gum tragacanth naturally-occurring phosphatides (e.g., soy bean, lecithin, esters or partial esters derived from fatty acids and hexitol), anhydrides (e.g., sorbitan monooleate), or condensation products of partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate).
  • the oily suspensions or emulsions can optionally contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • sweetening agents and flavoring agents can optionally be added to provide palatable oral preparations.
  • these compositions can be preserved by optionally adding an anti-oxidant, such as ascorbic acid.
  • exemplary acceptable vehicles and solvents useful in the compositions of this disclosure is water, Ringer's solution, or isotonic sodium chloride solution.
  • sterile, fixed oils may be employed as a solvent or suspending medium for the dsRNA of this disclosure.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of parenteral formulations.
  • compositions and methods that feature the presence or administration of one or more dsRNA or analogs thereof of this disclosure, combined, complexed, or conjugated with a polypeptide, optionally formulated with a pharmaceutically-acceptable carrier, such as a diluent, stabilizer, buffer, or the like.
  • a pharmaceutically-acceptable carrier such as a diluent, stabilizer, buffer, or the like.
  • the negatively charged dsRNA molecules of this disclosure may be administered to a patient by any standard means, with or without stabilizers, buffers, or the like, to form a composition suitable for treatment.
  • standard protocols for formation of liposomes can be followed.
  • compositions of the present disclosure may also be formulated and used as a tablet, capsule or elixir for oral administration, suppository for rectal administration, sterile solution, or suspension for injectable administration, either with or without other compounds known in the art.
  • dsRNAs of the present disclosure may be administered in any form, such as nasally, transdermally, parenterally, or by local injection.
  • compositions thereof, and methods of the present disclosure include those suffering from one or more disease or condition mediated, at least in part, by overexpression or inappropriate expression of a STAT3 gene, or which are amenable to treatment by reducing expression of a STAT3 protein, including one or more hyperproliferative disorders, for example leukemia, lymphoma, breast cancer, prostate cancer, multiple myeloma, and head and neck cancer, as well as tumor proliferation.
  • the compositions and methods of this disclosure are also useful as therapeutic tools to regulate expression of STAT3 to treat or prevent symptoms of, for example, the conditions listed herein.
  • dsRNA or substituted or modified dsRNA, as described herein, comprising a first strand that is complementary to a human STAT3 mRNA as set forth in SEQ ID NO: 1158, 1159, or 1160, and a second strand and a third strand that is each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein the mdRNA molecule optionally includes at least one double-stranded region comprises from 5 base pairs to 13 base pairs.
  • subjects can be effectively treated, prophylactically or therapeutically, by administering an effective amount of one or more dsRNA having a first strand that is complementary to a human STAT3 mRNA as set forth in SEQ ID NO: 1158, 1159, or 1160, and a second strand and a third strand that is each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and the mdRNA molecule optionally includes at least one double-stranded region comprises from 5 base pairs to 13 base pairs and at least one pyrimidine of the mdRNA is substituted with a pyrimidine nucleoside according to Formula I or II:
  • At least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -OH, or R 1 is methyl, R 2 is -OH, and R 8 is S. In certain embodiments, at least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -O-methyl, or R 1 is methyl, R 2 is -O-methyl, and R 8 is O. In other embodiments, at least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -O-methyl, or R 1 is methyl, R 2 is -O-methyl, and R 8 is O.
  • a dsRNA will have from one to all uridines substituted with 5-methyluridine and have up to about 75% 2'-methoxy substitutions provided the 2'-methoxy substitutions are not at the Argonaute cleavage site.
  • a dsRNA will have from one to all uridines substituted with 5-methyluridine and have up to about 100% 2'-fluoro substitutions.
  • a dsRNA will have from one to all uridines substituted with 5-methyluridine and have up to about 75% 2'-deoxy substitutions.
  • a dsRNA will have up to about 75% LNA substitutions and have up to about 75% 2'-methoxy substitutions. In still other embodiments, a dsRNA will have up to about 75% LNA substitutions and have up to about 100% 2'-fluoro substitutions. In further exemplary methods, a dsRNA will have up to about 75% LNA substitutions and have up to about 75% 2'-deoxy substitutions. In further exemplary methods, a dsRNA will have up to about 75% 2'-methoxy substitutions and have up to about 100% 2'-fluoro substitutions.
  • a dsRNA will have up to about 75% 2'-methoxy substitutions and have up to about 75% 2'-deoxy substitutions. In further embodiments, a dsRNA will have up to about 100% 2'-fluoro substitutions and have up to about 75% 2'-deoxy substitutions.
  • a dsRNA will have from one to all uridines substituted with 5-methyluridine, up to about 75% LNA substitutions, and up to about 75% 2'-methoxy substitutions. In still further exemplary methods, a dsRNA will have from one to all uridines substituted with 5-methyluridine, up to about 75% LNA substitutions, and up to about 100% 2'-fluoro substitutions. In further exemplary methods, a dsRNA will have from one to all uridines substituted with 5-methyluridine, up to about 75% LNA substitutions, and up to about 75% 2'-deoxy substitutions.
  • a dsRNA will have from one to all uridines substituted with 5-methyluridine, up to about 75% 2'-methoxy substitutions, and up to about 75% 2'-fluoro substitutions. In further exemplary methods, a dsRNA will have from one to all uridines substituted with 5-methyluridine, up to about 75% 2'-methoxy substitutions, and up to about 75% 2'-deoxy substitutions. In further exemplary methods, a dsRNA will have from one to all uridines substituted with 5- methyluridine, up to about 100% 2'-fluoro substitutions, and up to about 75% 2'-deoxy substitutions.
  • a dsRNA will have from one to all uridines substituted with 5-methyluridine, up to about 75% LNA substitutions, up to about 75% 2'-methoxy, up to about 100% 2'-fluoro, and up to about 75% 2'-deoxy substitutions. In other exemplary methods, a dsRNA will have up to about 75% LNA substitutions, up to about 75% 2'-methoxy substitutions, and up to about 100% 2'-fluoro substitutions. In further exemplary methods, a dsRNA will have up to about 75% LNA substitutions, up to about 75% 2'-methoxy substitutions, and up to about 75% 2'-deoxy substitutions.
  • a dsRNA will have up to about 75% LNA substitutions, up to about 100% 2'-fluoro substitutions, and up to about 75% 2'-deoxy substitutions. In still further exemplary methods, a dsRNA will have up to about 75% 2'-methoxy, up to about 100% 2'-fluoro, and up to about 75% 2'-deoxy substitutions.
  • the dsRNA may further comprise up to 100% phosphorothioate internucleoside linkages, from one to ten or more inverted base terminal caps, or any combination thereof. Additionally, any of these multiple modification embodiments may have these multiple modifications on one strand, two strands, three strands, a plurality of strands, or all strands. Finally, in any of these multiple modification dsRNA, the dsRNA must have gene silencing activity.
  • At least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -OH, or R 1 is methyl, R 2 is -OH, and R 8 is S. In certain embodiments, at least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -O-methyl, or R 1 is methyl, R 2 is -O-methyl, and R 8 is O. In certain embodiments, at least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -O-methyl, or R 1 is methyl, R 2 is -O-methyl, and R 8 is O. In other embodiments, the internucleoside linking group covalently links from about 5 to about 40 nucleosides.
  • adjunctive therapeutic agents in these combinatorial formulations and coordinate treatment methods include, for example, enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules and other organic or inorganic compounds including metals, salts and ions, and other drugs and active agents indicated for treating a ST AT3 -associated disease or condition, including chemotherapeutic agents used to treat cancer, steroids, non-steroidal anti-inflammatory drugs (NSAIDs), tyrosine kinase inhibitors, or the like.
  • chemotherapeutic agents used to treat cancer steroids, non-steroidal anti-inflammatory drugs (NSAIDs), tyrosine kinase inhibitors, or the like.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • chemotherapeutic agents include alkylating agents (e.g., cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogen mustards, uramustine, temozolomide), antimetabolites (e.g., aminopterin, methotrexate, mercaptopurine, fluorouracil, cytarabine), taxanes (e.g., paclitaxel, docetaxel), anthracyc lines (e.g., doxorubicin, daunorubicin, epirubicin, idaruicin, mitoxantrone, valrubicin), bleomycin, mytomycin, actinomycin, hydroxyurea, topoisomerase inhibitors (e.g., camptothecin, topotecan, irinotecan, etoposide, teniposide), monoclonal antibodies (e.g., alemtuzuma
  • STAT3 or affect specific STAT3 biological activities.
  • inhibitors of STAT3 have been described that may be suitably employed as adjunctive therapies including, but not limited to, inhibiting proteins, small molecules, and antibodies or fragments thereof.
  • Inhibiting proteins include, for example, nuclear tyrosine phosphatase, SH2-containing phosphatase- 1, protein tyrosine phosphatase- IB, protein inhibitors of activated STATs (PIAs), as well as the suppressor of cytokine signaling (SOCS) family of proteins.
  • PIAs protein inhibitors of activated STATs
  • SOCS suppressor of cytokine signaling family of proteins.
  • the PIAs interact directly with STATs and block their DNA-binding activity.
  • the SOCS family of proteins bind to receptors and JAK family kinases and inhibit STAT activation and also act as STAT -induced STAT inhibitors (Adamkova et al, Folia Biologica 55:1, 2007).
  • Small molecule STAT3 inhibitors include, for example, resveratrol, silibinin, cucurbitactin Q, STA-21, TG101209, and Stahlic.
  • Resveratrol inhibits src tyrosine kinase activity and thereby blocks constitutive STAT3 protein activation in malignant cells (Kotha et al, MoI. Cancer Ther. 5:3, 2006).
  • Silibinin reduces constitutive STAT3 phosphorylation and was found to induce apoptosis in DU145 cells (Agarwal et al, Carcinogenesis 28:1, 2007).
  • Cucurbitactin Q inhibits the activation of STAT3 and induces apoptosis without inhibiting JAK2, Crc, Akt, Erk, or JNK (Sun et al, Oncogene 24, 2005).
  • STA-21 inhibits STAT3 dimerization, DNA binding, and nucleus translocation (Song et al, PNAS 102:13, 2005).
  • TGl 01209 inhibits JAK2 which, in turn, has been shown to inhibit STAT3 by not phosphorylating STAT3 (Pardanani et al, Leukemia, 20 September 2007).
  • a dsRNA is administered, simultaneously or sequentially, in a coordinated treatment protocol with one or more of the secondary or adjunctive therapeutic agents contemplated herein.
  • the coordinate administration may be done in any order, and there may be a time period while only one or both (or all) active therapeutic agents, individually or collectively, exert their biological activities.
  • a distinguishing aspect of all such coordinate treatment methods is that the dsRNA present in a composition elicits some favorable clinical response, which may or may not be in conjunction with a secondary clinical response provided by the secondary therapeutic agent.
  • a dsRNA of this disclosure can include a conjugate member on one or more of the terminal nucleotides of a dsRNA.
  • the conjugate member can be, for example, a lipophile, a terpene, a protein binding agent, a vitamin, a carbohydrate, or a peptide.
  • the conjugate member can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, or a C5 pyrimidine linker.
  • the conjugate member is a glyceride lipid conjugate (e.g., a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols.
  • Additional conjugate members include peptides that function, when conjugated to a modified dsRNA of this disclosure, to facilitate delivery of the dsRNA into a target cell, or otherwise enhance delivery, stability, or activity of the dsRNA when contacted with a biological sample (e.g., a target cell expressing STAT3).
  • Exemplary peptide conjugate members for use within these aspects of this disclosure include peptides PN27, PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN183, PN202, PN204, PN250, PN361, PN365, PN404, PN453, PN509, and PN963, described, for example, in U.S. Patent Application Publication Nos. 2006/0040882 and 2006/0014289, and U.S. Provisional Patent Application Nos. 60/822,896 and 60/939,578; and PCT Application PCT/US2007/075744, which are all incorporated herein by reference.
  • a dsRNA or analog thereof of this disclosure may be conjugated to the polypeptide and admixed with one or more non-cationic lipids or a combination of a non-cationic lipid and a cationic lipid to form a composition that enhances intracellular delivery of the dsRNA as compared to delivery resulting from contacting the target cells with a naked dsRNA.
  • the mixture, complex or conjugate comprising a dsRNA and a polypeptide can be optionally combined with (e.g., admixed or complexed with) a cationic lipid, such as LipofectineTM.
  • a cationic lipid such as LipofectineTM.
  • the dsRNA and peptide may be mixed together first in a suitable medium such as a cell culture medium, after which the cationic lipid is added to the mixture to form a dsRNA/delivery peptide/cationic lipid composition.
  • the peptide and cationic lipid can be mixed together first in a suitable medium such as a cell culture medium, followed by the addition of the dsRNA to form the dsRNA/delivery peptide/cationic lipid composition.
  • dsRNA compositions comprising surface-modified liposomes containing, for example, poly(ethylene glycol) lipids (PEG- modified, or long-circulating liposomes or stealth liposomes)
  • PEG- modified, or long-circulating liposomes or stealth liposomes PEG-modified, or stealth liposomes
  • compositions are provided for targeting dsRNA molecules of this disclosure to specific cell types, such as hepatocytes.
  • dsRNA can be complexed or conjugated glycoproteins or synthetic glycoconjugates glycoproteins or synthetic glycoconjugates having branched galactose (e.g., asialoorosomucoid), N-acetyl-D- galactosamine, or mannose (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429, 1987; Baenziger and Fiete, Cell 22: 611, 1980; Connolly et al., J. Biol. Chem. 257:939, 1982; Lee and Lee,
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence of, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the specific subject under consideration for treatment, concurrent medication, and other factors that those skilled in the medical arts will recognize. For example, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients may be administered depending on the potency of a dsRNA of this disclosure.
  • Dosage levels in the order of about 0.1 mg to about 140 mg per kilogram of body weight per day can be useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day).
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
  • Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
  • test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated, as compared to placebo- treated or other suitable control subjects.
  • Nucleic acid molecules and polypeptides can be administered to cells by a variety of methods known to those of skill in the art, including administration within formulations that comprise a dsRNA alone, a dsRNA and a polypeptide complex / conjugate alone, or that further comprise one or more additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, stabilizer, preservative, or the like.
  • additional components such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, stabilizer, preservative, or the like.
  • Other exemplary substances used to approximate physiological conditions include pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof.
  • conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • nucleic acid molecules such as the dsRNAs of this disclosure
  • Boado et al. J. Pharm. Sci. 57:1308, 1998; Tyler et al., FEBS Lett. 421:280, 1999; Pardridge et al, Proc. Nat' I Acad. Sci. USA 92:5592, 1995; Boado, Adv. Drug Delivery Rev. 15:73, 1995; Aldrian-Herrada et al., Nucleic Acids Res. 26:4910, 1998; Tyler et al., Proc. Nat'l Acad. Sci. USA £6:7053-7058, 1999; Akhtar et al.,
  • the dsRNA and nicked dsRNA are shown below and were synthesized using standard techniques.
  • the RISC activator influenza G 1498 dsRNA comprises a 21 nucleotide sense strand and a 21 nucleotide antisense strand, which can anneal to form a double-stranded region of 19 base pairs with a two deoxythymidine overhang on each strand.
  • firefly luciferase reporter activity was measured first by adding Dual- GloTM Luciferase Reagent (Promega, Madison, WI) for 10 minutes with shaking, and then quantitating the luminescent signal using a VICTOR 3 TM 1420 Multilabel Counter (PerkinElmer, Waltham, MA). After measuring the firefly luminescence, Stop & Glo ® Reagent (Promega, Madison, WI) was added for 10 minutes with shaking to simultaneously quench the firefly reaction and initiate the Renilla luciferase reaction, and then the Renilla luciferase luminescent signal was quantitated VICTOR 3 TM 1420 Multilabel Counter (PerkinElmer, Waltham, MA).
  • the influenza dicer substrate (G1498DS) nicked at any one of positions 8 to 14 was also highly active ( Figure 5). Phosphorylation of the 5 '-end of the 3 '-most strand of the nicked sense influenza sequence had essentially no effect on activity, but addition of a locked nucleic acid appears to improve activity.
  • the influenza dicer substrate dsRNA (G1498DS) was tested at 0.0004 nM, 0.002 nM, 0.005 nM, 0.019 nM, 0.067 nM, 0.233 nM, 0.816 nM, 2.8 nM, and 1OnM, while the mdRNA with a nick at position 13 (G1498DS:Nkdl3) was tested at 0.001 nM, 0.048 nM, 0.167 nM, 1 nM, 2 nM, 7 nM, and 25 nM (see Figure 6).
  • G indicates a locked nucleic acid G in the 5' sense strand.
  • Example 2 The dual fluorescence assay of Example 2 was used to measure knockdown activity. Similar results were obtained at both the 5 nM and 10 nM concentrations. These data show that an mdRNA having a gap of up to 6 nucleotides still has activity, although having four or fewer missing nucleotides shows the best activity (see, also, Figure 7). Thus, mdRNA having various sizes gaps that are in various different positions have knockdown activity.
  • A indicates a locked nucleic acid A in each sense strand.
  • Example 3 The dual fluorescence assay of Example 3 was used to measure knockdown activity. These data show that increasing the number of locked nucleic acid substitutions tends to increase activity of an mdRNA having a nick at any of a number of positions.
  • the single locked nucleic acid per sense strand appears to be most active when the nick is at position 11 (see Figure 9). But, multiple locked nucleic acids on each sense strand make mdRNA having a nick at any position as active as the most optimal nick position with a single substitution (i.e., position 11) ( Figure 9). Thus, mdRNA having duplex stabilizing modifications make mdRNA essentially equally active regardless of the nick position.
  • the mdRNA sequences have a nicked sense strand after position 12, 13, and 14, respectively, as counted from the 5'-end, and the G at position 2 is substituted with locked nucleic acid G.
  • Vero cells were seeded at 6.5 x 10 4 cells/well the day before transfection in 500 ⁇ l 10% FBS/DMEM media per well.
  • Samples of 100, 10, 1, 0.1, and 0.01 nM stock of each dsRNA were complexed with 1.0 ⁇ l (1 mg/ml stock) of LipofectamineTM 2000 (Invitrogen, Carlsbad, CA) and incubated for 20 minutes at room temperature in 150 ⁇ l OPTIMEM (total volume) (Gibco, Carlsbad, CA). Vero cells were washed with OPTIMEM, and 150 ⁇ l of the transfection complex in OPTIMEM was then added to each well containing 150 ⁇ l of OPTIMEM media. Triplicate wells were tested for each condition. An additional control well with no transfection condition was prepared. Three hours post transfection, the media was removed.
  • Each well was washed once with 200 ⁇ l PBS containing 0.3% BSA and 10 mM HEPES/PS.
  • Cells in each well were infected with WSN strain of influenza virus at an MOI 0.01 in 200 ⁇ l of infection media containing 0.3% BSA/10 mM HEPES/PS and 4 ⁇ g/ml trypsin.
  • the plate was incubated for 1 hour at 37°C. Unadsorbed virus was washed off with the 200 ⁇ l of infection media and discarded, then 400 ⁇ l DMEM containing 0.3% BSA/10 mM HEPES/PS and 4 ⁇ g/ml trypsin was added to each well.
  • TCID50 assays 50% Tissue-Culture Infective Dose, WHO protocol
  • titers were estimated using the Spearman and Karber formula. The results show that these mdRNAs show about a 50% to 60% viral titer knockdown, even at a concentration as low aslO pM ( Figure 11).
  • An in vivo influenza mouse model was also used to examine the activity of a dicer substrate nicked dsRNA in reducing influenza virus titer as compared to a wild-type dsRNA (i.e., not having a nick).
  • Female BALB/c mice (age 8-10 weeks with 5-10 mice per group) were dosed intranasally with 120 nmol/kg/day dsRNA (formulated in C12-norArg(NH 3 +Cr)- C12/DSPE-PEG2000/DSPC/cholesterol at a ratio of 30:1 :20:49) for three consecutive days before intranasal challenge with influenza strain PR8 (20 PFU/mouse).
  • TCID50 the viral titer
  • Bronchial lavage was performed with 0.5 mL ice-cold 0.3% BSA in saline two times for a total of 1 mL.
  • BALF was spun and supernatants collected and frozen until cytokine analysis.
  • Blood was collected from the vena cava immediately following euthanasia, placed into serum separator tubes, and allowed to clot at room temperature for at least 20 minutes. The samples were processed to serum, aliquoted into Millipore ULTRAFREE 0.22 ⁇ m filter tubes, spun at 12,000 rpm, frozen on dry ice, and then stored at -7O 0 C until analysis.
  • Cytokine analysis of BALF and plasma were performed using the ProcartaTM mouse 10-Plex Cytokine Assay Kit (Panomics, Fremont, CA) on a Bio-PlexTM array reader. Toxicity parameters were also measured, including body weights, prior to the first dose on day 0 and again on day 3 (just prior to euthanasia). Spleens were harvested and weighed (normalized to final body weight). The results are provided in Table 5.
  • the mdRNA (RISC or dicer sized) induced cytokines to lesser extent than the intact (i.e., not nicked) parent molecules.
  • the decrease in cytokine induction was greatest when looking at IL-12(p40), the cytokine with consistently the highest levels of induction of the 10 cytokine multiplex assay.
  • the decrease in IL- 12 (p40) ranges from 25- to 56-fold, while the reduction in either IL-6 or TNF ⁇ induction was more modest (the decrease in these two cytokines ranges from 2- to 10-fold).
  • the mdRNA structure appears to provide an advantage in vivo in that cytokine induction is minimized compared to unmodified dsRNA.

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Abstract

La présente invention concerne des molécules d'acide ribonucléique méroduplex (ARNmd) capables d'induire la réduction ou le silençage de l'expression du gène STAT3. Un ARNmd selon cette invention comporte au moins trois brins se combinant pour former au moins deux régions à double brin qui ne se chevauchent pas et qui sont séparées par une rupture ou une brèche, un brin étant complémentaire d'un ARNm de STAT3. De plus, le méroduplex peut présenter au moins une uridine qui est remplacée par une 5-méthyluridine, un nucléoside qui est remplacé par un acide nucléique bloqué, ou éventuellement d'autres modifications, et toute combinaison de celles-ci. Cette invention concerne également des procédés permettant de réduire l'expression d'un gène STAT3 dans une cellule ou chez un patient, afin de traiter une maladie liée à STAT3.
PCT/US2008/055606 2007-03-02 2008-03-03 Composés d'acides nucléiques conçus pour inhiber l'expression du gène stat3 et utilisations de ceux-ci WO2008109494A1 (fr)

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