WO2012021383A2

WO2012021383A2 - RNA INTERFERENCE MEDIATED INHIBITION OF MITOGEN-ACTIVATED PROTEIN KINASE 1 (MAPK1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)

Info

Publication number: WO2012021383A2
Application number: PCT/US2011/046678
Authority: WO
Inventors: Steven Bartz; Duncan Brown; Yong Ma; Jenia Semyonov
Original assignee: Merck Sharp & Dohme Corp.
Priority date: 2010-08-10
Filing date: 2011-08-05
Publication date: 2012-02-16
Also published as: WO2012021383A3

Abstract

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of MAPK1 gene expression and/or activity, and/or modulate a MAPK1 gene expression pathway. Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules that are capable of mediating or that mediate RNA interference (RNAi) against MAPK1 gene expression.

Description

R.N A INTERFERENCE MEDIATED INHIBITION OF MITOGEN-ACTIVATED PROTEIN KINASE 1 (MAPK1) GENE EXPRESSION USING

SHORT INTERFERING NUCLEIC ACID (siNA)

SEQUENCE LISTING

[0001] The sequence listing submitted via EFS, in compliance with 37 CFR § 1.52(e)(5), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file "SequenceListingSIRONC3", created on July 25, 2011 , which is 833,376 bytes in size.

BACKGROUND OF THE INVENTION

[0002] Studies on signal transduction pathways have generated various promising molecular targets for therapeutic inhibition in cancer therapy. The Ras/MAPK pathway represents an important pathway for such therapeutic targets. Constitutive activation of the Ras MAPK pathway has been shown to be driven by ligand-induced stimulation of receptor tyrosine kinases or by ligand-independent mechanisms. Ligand-independent mechanisms include increased expression of or activating mutations in RTKs and downstream components. Ras is the most frequently mutated oncogene in human cancers and is activated in approximately 30% of human tumors. Including mutations of components of the Ras/MAPK effector pathway the frequency nearly reaches 50%. Activating mutations in Ras and BRaf have been identified in several human cancers, including melanoma, colon, pancreatic, ovarian and lung cancer. Pre-chnical studies further support the importance of the Ras/MAPK pathway in cancer initiation and progression as activated mutants of Ras, Raf and MEK have been shown to induce transformation and tumorigenesis in several cell types.

[0003] MAP kinases are evolutionary conserved signaling molecules by which cells transduce extracellular signals into intracellular responses. The MAPK1 gene encodes the mitogen-activated protein kinase 1 (also called extracellular signal-regulated kinase 2 (Erk2)). MAPK1 and MAPK3 (Erkl) are 85% identical protein kinases with a molecular weight of 42 kDa and 44 kDa, respectively. MAPK1 and MAPK3 are ubiquitously expressed in mammalian cells and have mostly overlapping, but some non-overlapping functions. Targeted deletion studies in mouse model have shown that MAPK1 and MEK1 , but not MAPK3 and MEK2, are essential for embryonic development Sturgill et al, 2008 Biochem and Biophys Res Comm 371 : 1-4;.

[0004] MAPKl and MAPK3 are activated by mitogens, growth factors, differentiation stimuli and cytokines. Activation of RTKs by their respective ligands results in activation of the small GTPase Ras, which in turn activates the MAP kinase kinase kinase Raf. Activated Raf phosphorylates and activates the MAP kinase kinases MEK1 and MEK2, which ultimately leads to the activation of MAPKl and MAPK3. Activated MAPKl and MAPK3 translocate to the nucleus and phosphorylates substrates with different cellular function including other protein kinases, transcription factors and cytoskeletal proteins. Activation of MAPKl and MAPK3 is negatively regulated by a family of MAPK phosphatases.

[0005] Activated MAPKl and MAPK3 regulates cell proliferation and survival by regulating the activity of more than 100 substrates in the cytoplasm and nucleus (Mebratu et al. 2009 Cell Cycle, 8: 1 168-75;. Cytoplasmic MAPKl and MAPK3 activate the RSK (ribosomal S6 family kinases) family of serine/threonine kinases. Activated RSK translocates to the nucleus and phosphorylates the pro-apoptotic protein BAD as well as the transcription factor CREB (cAMP response element-binding protein) to promote cell survival. In addition, MAPKl and MAPK3 directly phosphorylate the pro-apoptotic protein BIM to target it for degradation. Nuclear MAPKl and MAPK3 affect the activity of several transcription factors that induce the expression of multiple genes required for cell proliferation and survival. Among other nuclear substrates, activated MAPKl and MAPK3 phosphorylate the ternary complex factor Elk-1 , which increases its affinity for the serum response factor (SRF) and enhances transcription of several growth related genes, including members of the Fos and Jun family. Furthermore, MAPKl and MAPK3 affect FOXO transcription factors that activate multiple target genes involved in tumor suppression including pro-apoptotic factors, such as BIM, and cell cycle regulators, such as p27 and cyclin D. Phosphorylation of FOX03a by MAPKl and MAPK3 results in enhanced degradation of FOX03a. MAPKl and MAPK3 also phosphorylate the oncogene c-Myc resulting in enhanced c-Myc protein stability (Meloche et al., 2007 Oncogene 26: 3227-39). Nuclear translocation of MAPKl and MAPK3 is regulated by PEA-15 (phosphoprotein enriched in astrocytes), whereas signaling to cytosolic substrates by MAPKl and MAPK3 is inhibited by Sef, which binds MEK/MAPK complexes. [0006] Multiple reports have shown that the Ras/MAPK pathway is constitutively activated in hepatocellular carcinoma (HCC). Mutations in Ras or BRaf are rare in HCC, but overexpression of CRaf has been reported in HCC patients fWhittaker et al, 2010 Oncogene Mar: 1-17). Increased activity of signaling intermediates, such as phosphorylated MEK1 and MEK2 and MAPKl and MAPK3, has been documented in up to 70% of HCC tumors compared with surrounding liver. Furthermore, the Ras/MAPK pathway can be activated through HBC and HCV infection, known aetiologies for HCC. MAPK l and MAPK3 activity correlates positively with tumor size and is associated with more aggressive HCC tumors. Hepatocellular carcinoma (HCC) is a common and aggressive cancer for which effective therapies are lacking.

[0007] Experiments using R Ai-mediated silencing have shown that silencing of MEK1 or MAPK l , but not MEK2 or MAPK3, abolish cell proliferation and DNA replication in vitro as well as tumor growth in vivo using the human HCC cell line Huh7 Gailhouste et al, 2010 Int J Cancer Mar. 126 (6): 1367-77).

[0008] In addition to its role in HCC, the Ras/MAPK pathway has been implicated in the progression of several types of human cancers including melanoma, colon, breast, thyroid and lung cancers (Montagut el al, 2009 Cancer Letters 283, 125-134 .

[0009] K-Ras mutations are observed in 90% pancreas, 45% colon and 35% of non-small cell lung cancer (NSCLC) patients. Expression of oncogenic K-Ras is sufficient to induce hyperplasia in preclinical mouse models including sarcomas, pulmonary, pancreatic and colonic neoplasms (Karreth et al, 2009 Current Opinion in Genetics & Dev Feb. 19:4-1 \). Even though N-Ras mutations are more prevalent in melanoma cancer patients (22%) than K-Ras mutations (2%), expression of mutated K-Ras in the melanocyte lineage results in the development of tumors with characteristics of human melanoma.

[0010] A number of reviews on the Ras/MAPK pathway and its role in cancer have been published: see for example Young et al, 2009 Adv Cancer Res 102: 1-17; Montagut et al, 2009 Cancer Letters 283: 125-134; Wong et al, 2009 Recent Pat Anticancer Drug Discovery 4: 28- 35; Schubbert et al, 2007 Nat Rev Cancer 7: 295-308; Roberts et al 2007 Oncogene, 26: 3291 - 310; Dhillon et al 2007 Oncogene, 26: 3279-92. Since dysregulation of the Ras/MAPK pathway has been implicated as a factor in tumorgenesis and disease progression in many cancers, different strategies for therapeutic inhibition of MAPK1 should be investigated. Specific inhibitors against MAPK1 gene expression have important therapeutic value for the treatment of cancers in which MAPK1 activity contributes to the invasive/metastatic phenotype. For this reason, there remains a need for molecules that inhibit MAPK1.

[0011] Alteration of gene expression, specifically APK1 gene expression, through RNA interference (hereinafter "RNAi") is one approacli for meeting this need. RNAi is induced by short single-stranded RNA ("ssRNA") or double-stranded RNA ("dsRNA") molecules. The short dsRNA molecules, called "short interfering nucleic acids ("siNA")" or "short interfering RNA" or "siRNA" or "RNAi inhibitors" silence the expression of messenger RNAs ("mRNAs") that share sequence homology to the siNA. This can occur via cleavage of the mRNA mediated by an endonuclease complex containing a siNA, commonly referred to as an RNA-induced silencing complex (RISC). Cleavage of the target RNA typically takes place in the middle of the region complementaiy to the guide sequence of the siNA duplex (Elbashir et al., 2001 , Genes Dev., 15: 188). In addition, RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably through cellular mechanisms that either inhibit translation or that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297: 1818-1819; Volpe et al., 2002, Science, 297: 1833-1837; Jenuwein, 2002, Science, 297:2215-2218; and Hall et al, 2002, Science, 297:2232-2237). Despite significant advances in the field of RNAi, there remains a need for agents that can inhibit MAPK1 gene expression and that can treat disease associated with MAPK1 expression such as cancer.

SUMMARY OF THE INVENTION

[0012] The invention provides a solution to the problem of treating diseases that respond to the modulation of the MAPK1 gene expression using novel short interfering nucleic acid (siNA) molecules to modulate MAPK1 expression.

[0013] The present invention provides compounds, compositions, and methods useful for modulating the expression of MAPK1 genes, specifically those MAPKl genes associated with cancer and for treating such conditions by RNA interference (RNAi) using small nucleic acid molecules. [0014] In particular, the instant invention features small nucleic acid molecules, i.e., short interfering nucleic acid (siNA) molecules including, but not limited to, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA) and circular RNA molecules and methods used to modulate the expression of MAPK1 genes and/or other genes involved in pathways of MAPK1 gene expression and/or activity.

[0015] In one aspect, the invention provides double-stranded short interfering nucleic acid (siNA) molecules that inhibit the expression of a MAPK1 gene in a cell or mammal, wherein the double-stranded siNAs comprise a sense and an antisense stand. The antisense strand comprises a sequence that is complementary to at least a part of an RNA associated with the expression of the MAP 1 gene. The sense strand comprises a sequence that is complementary to the antisense strand. In various embodiments, at least one strand comprises at least a 15 nucleotide sequence selected from the group of sequences consisting of SEQ ID NOS: l-1462. In certain embodiments, the antisense strand comprises at least 15 nucleotides having sequence complementarity to a target sequence set forth in Table la. In other and/or in the same embodiments, the antisense strand comprises at least a 15 nucleotide sequence of one of the antisense sequences set forth in Table lb. In some embodiments, the sense strand comprises at least a 1 nucleotide sequence of a sense strand sequence as set forth in Table lb.

[0016] In certain embodiments of this aspect of the invention, double-stranded short interfering nucleic acid (siNA) molecules are provided wherein the antisense stand comprises a modified sequence as set forth in Table lc that has sequence complementarity to a target sequence of the invention. In some embodiments, the sense strand also comprises a modified sequence as set forth in Table lc.

[0017] In certain embodiments, the present invention provides a double-stranded short interfering nucleic acid (siNA) molecule that modulates the expression of MAPK1 , wherein the siNA comprises a sense strand and an antisense strand; each strand is independently 15 to 30 nucleotides in length; and the antisense strand comprises at least 15 nucleotides having sequence complementary to any of:

5 '-AGAACAUCAUUGGAAUCAA-3' (SEQ ID NO: 1 );

5 '-UAUCCAUUCAGCUAACGUU-3' (SEQ ID NO: 2); 5'-UCCAUUCAGCUAACGUUCU-3' (SEQ ID NO: 3); or

5'-UUAUGAUAAUGUCAACAAA-3' (SEQ ID NO: 4).

[0018] In some embodiments of the invention, the antisense strand of a siNA molecule comprises at least a 15 nucleotide sequence of:

5'-UUGAUUCCAAUGAUGUUCU-3' (SEQ ID NO: 1210);

5'-AACGUUAGCUGAAUGGAUA-3^* (SEQ ID NO: 121 1);

5 ' - AG A ACGU U AGCUG A AUGG A-3 ' (SEQ ID NO: 1212); or 5'-UUUGUUGACAUUAUCAUAA-3' (SEQ ID NO: 1213);

[0019] In some embodiments, the sense strand of a siNA molecule of the invention comprises at least a 15 nucleotide sequence of:

5'-AGAACAUCAUUGGAAUCAA-3^* (SEQ ID NO: 1);

5'-UAUCCAUUCAGCUAACGUU-3' (SEQ ID NO: 2);

5'-UCCAUUCAGCUAACGUUCU-3' (SEQ ID NO: 3); or

5'-UUAUGAUAAUGUCAACAAA-3' (SEQ ID NO: 4).

[0020] In some embodiments, a siNA molecule of the invention comprises any of:

5'-AGAACAUCAUUGGAAUCAA-3' (SEQ ID NO: 1) and 5'- UUGAUUCCAAUGAUGUUCU-3' (SEQ ID NO: 1210); or

5'-UAUCCAUUCAGCUAACGUU-3' (SEQ ID NO: 2); and 5'- AACGUU AGCUG A AUGG AU A-3' (SEQ ID NO : 121 1 ); or

5'-UCCAUUCAGCUAACGUUCU-3' (SEQ ID NO: 3) and 5'-

AGAACGUUAGCUGA AUGG A-3' (SEQ ID NO: 1212); or

5'-UUAUGAUAAUGUCAACAAA-3' (SEQ ID NO: 4); and 5'- UUUGUUGACAUUAUCAUAA-3' (SEQ ID NO: 1213). [0021] In some embodiments of the invention, all of the nucleotides of siNAs of the invention are unmodified. In other embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of the nucleotide positions independently in either one or both strands of an siNA molecule are modified. Modifications include nucleic acid sugar modifications, base modifications, backbone (intemucleotide linkage) modifications, non-nucleotide modifications, and/or any combination thereof. In certain instances, purine and pyrimidine nucleotides are differentially modified. For example, purine and pyrimidine nucleotides can be differentially modified at the 2'-sugar position (i.e., at least one purine has a different modification from at least one pyrimidine in the same or different strand at the 2'-sugar position). In certain instances the purines are unmodified in one or both strands, while the pyrimidines in one or both strands are modified. In certain other instances, the pyrimidines are unmodified in one or both strands, while the purines in one or both strands are modified. In some instances, at least one modified nucleotide is a 2'-deoxy-2'-fjuoro nucleotide, a 2'-deoxy nucleotide, or a 2'-0-alkyl nucleotide. In some instances, at least 5 or more of the pyrimidine nucleotides in one or both stands are either all 2'-deoxy-2'-fluoro or all 2'-0-methyl pyrimidine nucleotides. In some instances, at least 5 or more of the purine nucleotides in one or both stands are either all 2'-deoxy-2'-fluoro or all 2'-0-methyl purine nucleotides. In certain instances, wherein the siNA molecules comprise one or more modifications as described herein, the nucleotides at positions 1 , 2. and 3 at the 5' end of the guide (antisense) strand are unmodified.

[0022] In certain embodiments, the siNA molecules of the invention have 3' overhangs of one, two, three, or four nucleotide(s) on one or both of the strands. In other embodiments, the siNA molecules lack overhangs (i.e., have blunt ends). Preferably, the siNA molecule has 3' overhangs of two nucleotides on both the sense and antisense strands. The overhangs can be modified or unmodified. Examples of modified nucleotides in the overhangs include, but are not limited to, 2'-0-alkyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, locked nucleic acid (LNA) nucleotides, or 2'-deoxy nucleotides. The overhang nucleotides in the antisense strand can comprise nucleotides that are complementary to nucleotides in the MAPK1 target sequence. Likewise, the overhangs in the sense stand can comprise nucleotides that are in the MAP l target sequence. In certain instances, the siNA molecules of the invention have two 3' overhang nucleotides on the antisense stand that are 2'-0-alkyl (e.g., 2'-0-methyl) nucleotides and two 3' overhang nucleotides on the sense stand that are 2'-deoxy nucleotides. In other instances, the siNA molecules of the invention have two 3' overhang nucleotides that are 2'-0-alkyl (e.g., 2'-0- methyl) nucleotides on both the antisense stand and on the sense stand. In certain embodiments, the 2'-0-alkyl nucleotides are 2'-0-methyl uridine nucleotides. In certain instances, the overhangs also comprise one or more phosphorothioate linkages between nucleotides of the overhang.

[0023] In some embodiments, the siNA molecules of the invention have caps (also referred to herein as "terminal caps." The cap can be present at the 5'-terminus (5'-cap) or at the 3 - terminus (3'-cap) or can be present on both termini, such as at the 5' and 3' termini of the sense strand of the siNA.

[0024] In some embodiments, the siNA molecules of the invention are phosphorylated at the 5' end of the antisense strand. The phosphate group can be a phosphate, a diphosphate or a triphosphate.

[0025] The siNA molecules of the invention when double stranded can be symmetric or asymmetric. Each strand of these double stranded siNAs independently can range in nucleotide length between 3 and 30 nucleotides. Generally, each strand of the siNA molecules of the invention is about 15 to 30 (i.e., about 19, 20, 21 , 22, 23 or 24) nucleotides in length.

[0026] The siNA molecules of the invention, which are double stranded or have a duplex structure, independently comprise about 3 to about 30 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs. Generally, the duplex structure of siNAs of the invention is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length.

[0027] In certain embodiments, double-stranded short interfering nucleic acid (siNA) molecules are provided, wherein the molecule has a sense strand and an antisense strand and comprises formula (A): B

B (N)_X [ ]χ5 -5'

(A) wherein, the upper strand is the sense strand and the lower strand is the antisense strand of the double-stranded nucleic acid molecule; wherein the antisense strand comprises at least a 15 nucleotide sequence of SEQ ID NO: 1210, SEQ ID NO: 121 1, SEQ ID NO: 1212, or SEQ ID NO: 1213, and the sense strand comprises a sequence having complementarity to the antisense strand; each N is independently a nucleotide which is unmodified or chemically modified or a non-nucleotide; each B is a terminal cap that is present or absent;

(N) represents overhanging nucleotides, each of which is independently unmodified or chemically modified;

[N] represents nucleotides that are ribonucleotides; XI and X2 are independently integers from 0 to 4; X3 is an integer from 15 to 30; X4 is an integer from 9 to 30; and

X5 is an integer from 0 to 6, provided that the sum of X4 and X5 is 15-30.

[0028] In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) of formula (A); wherein

(a) one or more pyrimidine nucleotides in Νχ positions are independently 2'-deoxy- 2'-fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof;

(b) one or more purine nucleotides in Νχ₄ positions are independently 2'-deoxy-2'- fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof; (c) one or more pyrimidine nucleotides in Νχ₃ positions are independently 2'-deoxy- 2'-fIuoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof; and

(d) one or more purine nucleotides in Νχ₃ positions are independently 2'-deoxy-2'- fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides,

[0029] The present invention further provides compositions comprising the double-stranded nucleic acid molecules described herein with optionally a pharmaceutically acceptable carrier or diluent.

[0030] The administration of the composition can be carried out by known methods, wherein the nucleic acid is introduced into a desired target cell in vitro or in vivo.

[0031] Commonly used techniques for introduction of the nucleic acid molecules of the invention into cells, tissues, and organisms include the use of various carrier systems, reagents and vectors. Non-limiting examples of such carrier systems suitable for use in the present invention include conjugates, nucleic-acid-lipid particles, lipid nanoparticles (LNP), liposomes, lipoplexes, micelles, virosomes, virus like particles (VLP), nucleic acid complexes, and mixtures thereof.

[0032] The compositions of the invention can be in the form of an aerosol, dispersion, solution (e.g., an injectable solution), a cream, ointment, tablet, powder, suspension or the like. These compositions may be administered in any suitable way, e.g. orally, sublingually, buccally, parenterally, nasally, or topically. In some embodiments, the compositions are aerosolized and delivered via inhalation.

[0033] The molecules and compositions of the present invention have utility over a broad range of therapeutic applications. Accordingly another aspect of this invention relates to the use of the compounds and compositions of the invention in treating a subject. The invention thus provides a method for treating a subject, such as a human, suffering from a condition which is mediated by the action, or by the loss of action, of MAPKl , wherein the method comprises administering to the subject an effective amount of a double-stranded short interfering nucleic acid (siNA) molecule of the invention. In certain embodiments, the condition is cancer. [0034] These and other aspects of the invention will be apparent upon reference to the following detailed description and attached figures. Moreover, it is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0035] Additionally, patents, patent applications, and other documents are cited throughout the specification to describe and more specifically set forth various aspects of this invention. Each of these references cited herein is hereby incorporated by reference in its entirety, including the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA- dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms that recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.

[0037] Figure 2 shows non-limiting examples of chemically modified siNA constructs of the present invention using a generalized structure of a representative siNA duplex. The specific modifications shown in the figure can be utilized alone or in combination with other modifications of the figure, in addition to other modifications and features described herein with reference to any siNA molecule of the invention. In the figure, N stands for any nucleotide or optionally a non-nucleotide as described here. The upper strand, having Β-Νχ₃-(Ν)χ₂-Β -3' is the sense (or passenger) strand of the siNA, whereas the lower strand, having Β(Ν)χι-Νχ₄-[Ν]χ5 -5' is the antisense (or guide) strand of the siNA. Nucleotides (or optional non-nucleotides) of internal portions of the sense strand are designated Νχ₃ and nucleotides (or optional non- nucleotides) of internal portions of the antisense strand are designated Νχ₄. Nucleotides (or optional non-nucleotides) of the internal portions are generally base paired between the two strands, but can optionally lack base pairing (e.g. have mismatches or gaps) in some embodiments. Nucleotides (or optional non-nucleotides) of overhang regions are designated by parenthesis (N). Nucleotides of the 5'-terminal portion of the antisense strand are designated [N]. Terminal caps are optionally present at the 5' and/or 3' end of the sense strand and further optionally present at the 3'-end of the antisense strand. Generally, each strand can independently range from about 15 to about 30 nucleotides in length, but can vary depending on the presence of any overhang nucleotides. In certain embodiments, X 1 and X2 are independently integers from 0 to 4; X3 is an integer from 15 to 30; X4 is an integer from 9 to 30; X5 is an integer from 0 to 6, provided that the sum of X4 and X5 is 15-30. Various modifications are shown for the nucleotides of the sense and antisense strands of the siNA constructs. The ( ) overhang nucleotide positions can be chemically modified as described herein (e.g., 2'-0-methyl, 2'- deoxy-2'-fluoro, 2'-deoxy, LNA, universal bases etc.) and can be either derived from a corresponding target nucleic acid sequence or not. The constructs shown in the figure can also comprise phosphorothioate linkages as described herein. For example, phosphorothioate linkages can exist between any N, (N), and/or [N] positions. Such phosphorothioate incorporation can be utilized between purine "R" and pyrimidine "Y" positions, or for stabilization of pyrimidine linkages in general. Furthermore, although not depicted on the Figure, the constructs shown in the figure can optionally include a ribonucleotide at the 9^th position from the 5'-end of the sense strand or the 1 1^th position based on the 5'-end of the guide strand by counting 1 1 nucleotide positions in from the 5'-terminus of the guide strand. Similarly, the antisense strand can include a ribonucleotide at the 14^th position from the 5'-end, or alternately can be selected or designed so that a 2'-0-alkyl nucleotide (e.g., a 2'-0-methyl purine) is not present at this position. Furthermore, although not shown in the Figure, the 5'-terminal position of the antisense strand can comprise a terminal phosphate group as described herein. The antisense strand generally comprises sequence complementary to any target nucleic acid sequence of the invention, such as those set forth in Table la herein.

[0038] Figure 3 shows non-limiting examples of certain combinations of modifications applied to the representative siNA duplex described in Figure 2. The table shown below the representative structure provides specific combinations of (Ν)χι, (Ν)χ2, Νχ , Νχ₄, and/or [Ν]χ₅ nucleotide (and optional non-nucleotide) positions. For example, combinations of 5 or more (e.g., 5, 6, 7, 8, 9, or 10 or more) Νχ₃ and 5 or more (e.g., 5, 6, 7, 8, 9, or 10 or more) Νχ₄ pyrimidine "Y" and purine "R" nucleotides are specified, each of which can independently have specific (Ν)χι, and/or (Ν)χ₂, substitutions as shown in the figure, in addition to optional phosphorothioate substitutions. The 5'-terminal antisense strand [N] nucleotides are generally ribonucleotides, but can also be modified or unmodified depending on if they are purine "R" or pyrimidine "Y" nucleotides

[0039] Figure 4A-C shows non-limiting examples of different siNA constructs of the invention. The criteria of the representative structures shown in Figures 2 and 3 can be applied to any of the structures shown in Figure 4A-C.

[0040] The examples shown in Figure 4A (constructs 1 , 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example, comprising about 1 , 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for R Ai activity. Construct 2 can comprise a polynucleotide or non- nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or i vitro.

[0041] The examples shown in Figure 4B represent different variations of double-stranded nucleic acid molecule of the invention, such as microR A, that can include overhangs, bulges, loops, and stem-loops resulting from partial complementarity. Such motifs having bulges, loops, and stem-loops are generally characteristics of miRNA. The bulges, loops, and stem-loops can result from any degree of partial complementarity, such as mismatches or bulges of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in one or both strands of the double-stranded nucleic acid molecule of the invention. [0042] The example shown in Figure 4C represents a model double-stranded nucleic acid molecule of the invention comprising a 19 base pair duplex of two 21 nucleotide sequences having dinucleotide 3'-overhangs. The top strand ( 1 ) represents the sense strand (passenger strand), the middle strand (2) represents the antisense (guide strand), and the lower strand (3) represents a target polynucleotide sequence. The dinucleotide overhangs (NN) can comprise a sequence derived from the target polynucleotide. For example, the 3'-(NN) sequence in the guide strand can be complementary to the 5'-[NN] sequence of the target polynucleotide. In addition, the 5'-(NN) sequence of the passenger strand can comprise the same sequence as the 5'-[NN] sequence of the target polynucleotide sequence. In other embodiments, the overhangs (NN) are not derived from the target polynucleotide sequence, for example where the 3'-(NN) sequence in the guide strand are not complementary to the 5'-[NN] sequence of the target polynucleotide and the 5'-(NN) sequence of the passenger strand can comprise different sequence from the 5'-[NN] sequence of the target polynucleotide sequence. In additional embodiments, any (NN) nucleotides are chemically modified, e.g., as 2'-0-methyl, 2'-deoxy-2'- fluoro, and/or other modifications herein. Furthermore, the passenger strand can comprise a ribonucleotide position N of the passenger strand. For the representative 19 base pair 21 mer duplex shown, position N can be 9 nucleotides in from the 3' end of the passenger strand. However, in duplexes of differing length, the position N is determined based on the 5'-end of the guide strand by counting 1 1 nucleotide positions in from the 5'-terminus of the guide strand and picking the corresponding base paired nucleotide in the passenger strand. Cleavage by Ago2 takes place between positions 10 and 1 1 as indicated by the arrow. In additional embodiments, there are two ribonucleotides. NN, at positions 10 and 1 1 based on the 5'-end of the guide strand by counting 10 and 3 1 nucleotide positions in from the 5'-terminus of the guide strand and picking the corresponding base paired nucleotides in the passenger strand.

[0043] Figure 5 shows non-limiting examples of different stabilization chemistries (1 -10) that can be used, for example, to stabilize the 5' and/or 3'-ends of siNA sequences of the invention, including (1) [3-3']-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5'-3']-3'- deoxyribonucleotide; (4) [5'-3']-ribonucleotide; (5) [5'-3']-3'-0-methyl ribonucleotide; (6) 3'- glyceryl; (7) [3'-5']-3'-deoxyribonucleotide; (8) [3'-3']~deoxyribonucleotide; (9) [5'-2']- deoxyribonucleotide; and (10) [5-3']-dideoxyribonucleotide (when X = O). In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different sugar and base nucleotide modifications as described herein.

[0044] Figure 6 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistant while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing 2'-modifications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct is tested in an appropriate system (e.g., human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay and/or against endogenous mRNA). Lead siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.

[0045] Figure 7 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.

[0046] Figure 8 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.

[0047] Figure 9 shows a non-limiting example of a cholesterol linked phosphoramidite that can be used to synthesize cholesterol conjugated siNA molecules of the invention. An example is shown with the cholesterol moiety linked to the 5'-end of the sense strand of an siNA molecule.

[0048] Figure 10 depicts an embodiment of 5' and 3' inverted abasic cap linked to a nucleic acid strand.

[0049] Figure 11 shows the results of an efficacy study in TRE-MET mice using MAP 1 siNA R-008274691-000S.

DETAILED DESCRIPTION OF THE INVENTION A. Terms and Definitions

[0050] The following terminology and definitions apply as used in the present application.

[0051] The term "abasic" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to sugar moieties lacking a nucleobase or having a hydrogen atom (H) or other non-nucleobase chemical groups in place of a nucleobase at the 1 ' position of the sugar moiety, see for example Adamic et al, U.S. Pat. No. 5,998,203. In one embodiment, an abasic moiety of the invention is a ribose, deoxyribose, or dideoxyribose sugar.

[0052] The term "acyclic nucleotide" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbon/carbon or carbon/oxygen bonds are independently or in combination absent from the nucleotide.

[0053] The term "alkyl" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a saturated or unsaturated hydrocarbons, including straight- chain, branched-chain, alkenyl, alkynyl groups and cyclic groups, but excludes aromatic groups. Notwithstanding the foregoing, alkyl also refers to non-aromatic heterocyclic groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted, the substituted group(s) is preferably, hydroxyl, halogen, cyano, C1 -C4 alkoxy, =0, =S, N0₂ , SH, NH₂, or NRiR₂, where R_x and R₂ independently are H or C1 -C4 alkyl.

[0054] The phrase "agents that interfere with cell cycle checkpoints" refers to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents.

[0055] The phrase "agents that interfere with receptor tyrosine kinases (RTKs)" refers to compounds that inhibit RTKs and therefore inhibit mechanisms involved in oncogenesis and tumor progression.

[0056] The phrase "androgen receptor modulators" refers to compounds that interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. [0057] The phrase "angiogenesis inhibitors" refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism.

[0058] The term "aryl" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaiyl groups, all of which can be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, C1 -C4 alkoxy, C 1 -C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, NH₂, and RiR₂ groups, where Rj and R₂ independently are H or C 1-C4 alkyl.

[0059] The term "alkylaryl" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and examples of heterocyclic aryl groups having such heteroatoms include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. Preferably, the alkyl group is a C 1-C4 alkyl group.

[0060] The term "amide" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to an -C(0)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen.

[0061] The phrase "antisense region" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to a nucleotide sequence of an siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of an siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule. In one embodiment, the antisense region of the siNA molecule is referred to as the antisense strand or guide strand. [0062] The phrase "asymmetric hairpin" refers to a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and fonn a duplex with loop. For example, an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system {e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprising about 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12) nucleotides, and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also comprise a 5'-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.

[0063] The term "biodegradable" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation.

[0064] The term "biodegradable linker" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to a linker molecule that is designed to connect one molecule to another molecule, and which is susceptible to degradation in a biological system. The linker can be a nucleic acid or non-nucleic acid based linker. For example, a biodegradable linker can be used to attach a ligand or biologically active molecule to an siNA molecule of the invention. Alternately, a biodegradable linker can be used to connect the sense and antisense strands of an siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, such as 2'-0-methyl, 2'-fluoro, 2'-amino, 2'-0-amino, 2'-C-allyl, 2'-0-allyl, and other 2'-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

[0065] The phrase "biologically active molecule" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system and/or are capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules. Examples of biologically active molecules, include siNA molecules alone or in combination with other molecules including, but not limited to therapeutically active molecules such as antibodies, cholesterol, hoimones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, polyamines, polyamides, polyethylene glycol, other polyethers, 2-5A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof.

[0066] The phrase "biological system" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to material, in a purified or unpurified form, from biological sources including, but not limited to, human or animal, wherein the system comprises the components required for RNAi activity. Thus, the phrase includes, for example, a cell, tissue, subject, or organism, or extract thereof. The term also includes reconstituted material from a biological source.

[0067] The phrase "blunt end" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to termini of a double-stranded siNA molecule having no overhanging nucleotides. For example, the two strands of a double-stranded siNA molecule having blunt ends align with each other with matched base-pairs without overhanging nucleotides at the termini. A siNA duplex molecule of the invention can comprise blunt ends at one or both termini of the duplex, such as termini located at the 5'-end of the antisense strand, the 5'-end of the sense strand, or both termini of the duplex.

[0068] The term "cap" also referred to herein as "terminal cap," as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to a moiety, which can be a chemically modified nucleotide or non-nucleotide that can be incorporated at one or more termini of one or more nucleic acid molecules of the invention. These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap) or at the 3'-terminal (3'-cap) or can be present on both termini of any nucleic acid molecule of the invention. A cap can be present at the 5'-end, 3-end and/or 5' and 3'-ends of the sense strand of a nucleic acid molecule of the invention. Additionally, a cap can optionally be present at the 3'-end of the antisense strand of a nucleic acid molecule of the invention. In non-limiting examples, the 5'-cap includes, but is not limited to, LNA; glyceryl; inverted deoxy abasic residue (moiety); 4',5 '-methylene nucleotide; l -(beta- D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitoI nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; t/ireo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4- dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide; 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; l,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety. Non-limiting examples of the 3'-cap include, but are not limited to, LNA; glyceryl; inverted deoxy abasic residue (moiety); 4', 5'-methylene nucleotide; l -(beta-D- erythrofuranosyl) nucleotide; 4'-thio nucleotide; carbocyclic nucleotide; 5'-amino-alkyl phosphate; l ,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; l ,2-aminododecyl phosphate; hydroxypropyl phosphate; l ,5-anhydrohexitol nucleotide; L- nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; t reo- pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5- dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'- phosphoramidate; 5'-phosphorothioate; l,4-butanediol phosphate; 5'-amino; bridging and/or non- bridging 5'-phosphoramidate; phosphorothioate and/or phosphorodithioate; bridging or non bridging methylphosphonate; and 5'-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein). Figure 5 shows some non- limiting examples of various caps.

[0069] The term "cell" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human being. The cell can be present in an organism, e.g. , birds, plants and mammals, such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g. , mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

[0070] The phrase "chemical modification" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to any modification of the chemical structure of the nucleotides that differs from nucleotides of native siR A or RNA in general. The term "chemical modification" encompasses the addition, substitution, or modification of native siRNA or RNA at the sugar, base, or internucleotide linkage, as described herein or as is otherwise known in the art. In certain embodiments, the term "chemical modification" can refer to certain forms of RNA that are naturally occurring in certain biological systems, for example 2'-0-methyl modifications or inosine modifications.

[0071] The term "complementarity" or "complementary" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplar nucleic acid molecules of the invention, the terms generally refer to the formation or existence of hydrogen bond(s) between one nucleic acid sequence and another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of bonding as described herein. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g. , Turner et al, 1 987, CSH Symp. Quant. Biol. LII pp.123- 133; Frier et al, 1986, Proc. Nat. Acad. Sci. USA 83 :9373-9377: Turner et al, 1987, J. Am. Chem. Soc. 109:3783-3785). Perfect complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Partial complementarity can include various mismatches or non-based paired nucleotides (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches, non-nucleotide linkers, or non-based paired nucleotides) within the nucleic acid molecule, which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the nucleic acid molecule or between the antisense strand or antisense region of the nucleic acid molecule and a corresponding target nucleic acid molecule. Such partial complementarity can be represented by a % complementarity that is determined by the number of non-base paired nucleotides, i.e., about 50%, 60%, 70%, 80%, 90% etc. depending on the total number of nucleotides involved. Such partial complementarity is permitted to the extent that the nucleic acid molecule (e.g., siNA) maintains its function, for example the ability to mediate sequence specific RNAi.

[0072] The terms "composition" or "formulation" as used herein refer to their generally accepted meaning in the art. These terms generally refer to a composition or formulation, such as in a pharmaceutically acceptable carrier or diluent, in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including, for example, a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, inhalation, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect. As used herein, pharmaceutical formulations include formulations for human and veterinary use. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: Lipid Nanoparticles (see for example Semple et al, 2010, Nat Biotechnol , Feb;28(2): 172-6.); P- glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL- lactide-coglycolide) microspheres for sustained release delivery (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al, 1998, J. Pharm. Sci, 87, 1308-1315; Tyler et al, 3999, FEBS Lett, 421 , 280-284; Pardridge et al, 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al, 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al, 1999, PNAS USA., 96, 7053-7058. A "pharmaceutically acceptable composition" or "pharmaceutically acceptable formulation" can refer to a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention to the physical location most suitable for their desired activity.

[0073J The phrase "cytotoxic/cytostatic agents" refer to compounds that cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell mytosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, inhibitors of histone deacetylase, inhibitors of kinases involved in mitotic progression, antimetabolites; biological response modifiers; hormonal/anti-honnonal therapeutic agents, hematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteasome inhibitors and ubiquitin ligase inhibitors.

[0074] The phrase "estrogen receptor modulators" refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism.

[0075] The term "gene" or "target gene" as used herein refers to their meaning as is generally accepted in the art. The terms generally refer a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide. The target gene can also include the UTR or non-coding region of the nucleic acid sequence. A gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (sfRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve as target nucleic acid molecules for siNA mediated RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Aberrant fRNA or ncRNA activity leading to disease can therefore be modulated by siNA molecules of the invention. siNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g. , transamination, methylation etc.). The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non- limiting examples of fungi include molds or yeasts. For a review, see for example Snyder and Gerstein, 2003, Science, 300, 258-260.

[0076] The phrase "HMG-CoA reductase inhibitors" refers to inhibitors of 3-hydroxy-3- methylglutaryl-CoA reductase. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds that have HMG-CoA reductase inhibitory activity, and therefore the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.

[0077] The phrase "homologous sequence" as used herein refers to its meaning as is generally accepted in the art. The term generally refers a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes. A homologous sequence can be a nucleotide sequence that is shared by two or more non- coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect identity (100%), as partially homologous sequences are also contemplated by and within the scope of the instant invention (e.g. , at least 95%, 94%, 93%, 92%, 91 %, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81 %, 80% etc.). Percent homology is the number of matching nucleotides between two sequences divided by the total length being compared, multiplied by 100.

[0078] The phrase "improved RNAi activity" refers to an increase in RNAi activity measured in vitro and/or in vivo, where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention. In this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all R A siNA or an siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced in vitro and/or in vivo.

[0079] The terms "inhibit," "down-regulate," or "reduce" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, he term generally refers the reduction in the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. Down-regulation can also be associated with post-transcriptional silencing, such as, RNAi mediated cleavage or by alteration in DNA methylation patterns or DNA chromatin structure. Inhibition, down-regulation or reduction with an siNA molecule can be in reference to an inactive molecule, an attenuated molecule, an siNA molecule with a scrambled sequence, or an siNA molecule with mismatches or alternatively, it can be in reference to the system in the absence of the nucleic acid.

[0080] The phrase "inhibitors of cell proliferation and survival signaling pathway" refers to pharmaceutical agents that inhibit cell surface receptors and signal transduction cascades downstream of those surface receptors.

[0081] The term "integrin blockers" refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the α_κβ₃ integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the _TOps integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the integrin and the α_Θβ₅ integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the α_Θβ,₆ <½β.8 αιβ.ι α₂βι 5βι α₆βι and αόβ₄ integrins. The term also refers to antagonists of any combination of α_σβ3, 0^5, α_Θβ₆ ^,β^ αι ι α₂βι ₅β.ι _έβι and α₆β integrins.

[0082] The terms "intermittent" or "intermittently" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to periodic stopping and starting at either regular or irregular intervals.

[0083] The terms "internucleoside linkage" or "internucleoside linker" or "internucleotide linkage" or "internucleotide linker" are used herein interchangeably and refer to any linker or linkage between two nucleoside units, as is known in the art, including, for example, but not limitation, phosphate, analogs of phosphate, phosphonate, guanidium, hydroxylamine, hydroxylhydrazinyl, amide, carbamate, alkyl, and substituted alkyl linkages. The internucleoside linkages constitute the backbone of a nucleic acid molecule.

[0084] The terms "mammalian" or "mammal" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to any warm blooded vertebrate species, such as a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the like.

[0085] The term "MAPKl" refers to mitogen-activated protein kinase 1 , which is gene that encodes MAPKl proteins, MAPKl peptides, MAPKl polypeptides, MAPKl regulatory polynucleotides (e.g., MAPKl miRNAs and siNAs), mutant MAPKl genes, and splice variants of a MAPKl genes, as well as other genes involved in MAPKl pathways of gene expression and/or activity. Thus, each of the embodiments described herein with reference to the term "MAPKl" are applicable to all of the protein, peptide, polypeptide, and/or polynucleotide molecules covered by the term "MAPKl", as that term is defined herein. Comprehensively, such gene targets are also referred to herein generally as "target" sequences (including the target sequences listed in Table la).

[0086] The phrase "metered dose inhaler" or MDI refers to a unit comprising a can, a secured cap covering the can and a formulation metering valve situated in the cap. MDI systems includes a suitable channeling device. Suitable channeling devices comprise for example, a valve actuator and a cylindrical or cone-like passage through which medicament can be delivered from the filled canister via the metering valve to the nose or mouth of a patient such as a mouthpiece actuator.

[0087] The term "microRNA" or "miRNA" as used herein refers to its meaning as is generally accepted in the art. The tenn generally refers a small double-stranded RNA that regulates the expression of target messenger RNAs either by mRNA cleavage, translational repression/inhibition or heterochromatic silencing (see for example Ambros, 2004, Nature, 431 , 350-355; Barrel, 2004, Cell, 1 16, 281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al, 2004, Nat. Rev. Genet., 5, 522-531 ; Ying el al, 2004, Gene, 342, 25-28; and Sethupathy et al, 2006, RNA, 12:192-197).

[0088] The term "modulate" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the tenn refers to when the expression of a gene, or level of one or more RNA molecules (coding or non-coding), or activity of one or more RNA molecules or proteins or protein subunits, is up-regulated or down-regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the molecule that effects modulation. For example, the term "modulate" in some embodiments can refer to inhibition and in other embodiments can refer to potentiation or up-regulation, e.g., of gene expression.

[0089] The phrase "modified nucleotide" as used herein refers to its meaning as is generally accepted in the art. The tenn generally refers a nucleotide, which contains a modification in the chemical structure of the base, sugar and/or phosphate of the unmodified (or natural) nucleotide as is generally known in the art. Non-limiting examples of modified nucleotides are described herein and in U.S. Application No. 12/064,014.

[0090] The phrase "NSAIDs that are selective COX -2 inhibitors" for purposes herein, refers to NSAIDs, which possess a specificity for inhibiting COX-2 over COX-1 of at least 100 fold as measured by the ratio of IC50 f°^r COX-2 over IC50 for COX-1 evaluated by cell or microsomal assays.

[0091] The phrase "non-base paired" refers to nucleotides that are not base paired between the sense strand or sense region and the antisense strand or antisense region of an double- stranded siNA molecule; and can include for example, but not limitation, mismatches, overhangs, single stranded loops, etc.

[0092] The term "non-nucleotide" refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, such as for example but not limitation abasic moieties or alkyl chains. The group or compound is "abasic" in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a nucleobase at the 1 '-position.

[0093] The term "nucleotide" is used as is generally recognized in the art. Nucleotides generally comprise a nucleobase, a sugar, and an internucleoside linkage, e.g., a phosphate. The base can be a natural bases (standard), modified bases, or a base analog, as are well known in the art. Such bases are generally located at the Γ position of a nucleotide sugar moiety. Additionally, the nucleotides can be unmodified or modified at the sugar, internucleoside linkage, and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others; see, for example, U.S. Application No. 12/064,014.

[0094] The term "overhang" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary double stranded nucleic acid molecules, the term generally refers to the terminal portion of a nucleotide sequence that is not base paired between the two strands of a double-stranded nucleic acid molecule (see for example, Figure 4). Overhangs, when present, are typically at the 3'-end of one or both strands in a siNA duplex.

[0095] The term "parenteral" as used herein refers to its meaning as is generally accepted in the art. The term generally refers methods or techniques of administering a molecule, drug, agent, or compound in a manner other than through the digestive tract, and includes epicutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.

[0096] The phrase "pathway target" refers to any target involved in pathways of gene expression or activity. For example, any given target can have related pathway targets that can include upstream, downstream, or modifier genes in a biologic pathway. These pathway target genes can provide additive or synergistic effects in the treatment of diseases, conditions, and traits herein.

[0097] The term "phosphorothioate" refers to an internucleotide phosphate linkage comprising one or more sulfur atoms in place of an oxygen atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

[0098] "Prenyl-protein transferase inhibitor" refers to a compound that inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-1), and geranylgeranyl-protein transferase type-II (GGPTase-Il, also called Rab GGPTase).

[0099] The phrase "retinoid receptor modulators" refers to compounds that interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism.

[0100] The term "ribonucleotide" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a nucleotide with a hydroxyl group at the 2' position of a β-D-ribofuranose moiety.

[0101] The term "RNA" as used herein refers to its generally accepted meaning in the art. Generally, the term RNA refers to a molecule comprising at least one ribofuranoside moiety. The term can include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0102] The phrase "RNA interference" or term "RNAi" refer to the biological process of inhibiting or down regulating gene expression in a cell, as is generally known in the art, and which is mediated by short interfering nucleic acid molecules, see for example Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamore et al, 2000, Cell, 101 , 25-33; Bass, 200L Nature, 41 1, 428-429; Elbashir et al., 2001 , Nature, 41 1 , 494-498; and Kreutzer et al. International PCT Publication No. WO 00/44895; Zernicka-Goetz et al, International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al, International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps- Depaillette, International PCT Publication No. WO 99/07409; and Li et al, International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al, 2002, Science, 297, 1833-3837; Jenuwein, 2002, Science, 297, 2215-221 8; and Hall et al, 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al, 2002, RNA, 8, 842-850; Reinhart et al, 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Battel, 2002, Science, 297, 1831 ). Additionally, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetics. For example, siNA molecules of the invention can be used to epigeneticaily silence genes at either the post- transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure or methylation patterns to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al, 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al, 2002, Science, 297, 1833- 1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al, 2002, Science, 297, 2232-2237). In another non-limiting example, modulation of gene expression by siNA molecules of the invention can result from siNA mediated cleavage of RNA (either coding or non-coding RNA) via RISC, or via translational inhibition, as is known in the art or modulation can result from transcriptional inhibition (see for example Janowski et al, 2005, Nature Chemical Biology, 1 , 216-222).

[0103] The phrase "'RNAi inhibitor" refers to any molecule that can down regulate, reduce or inhibit RNA interference function or activity in a cell or organism. An RNAi inhibitor can down regulate, reduce or inhibit RNAi (e.g., RNAi mediated cleavage of a target polynucleotide, translational inhibition, or transcriptional silencing) by interaction with or interfering with the function of any component of the RNAi pathway, including protein components such as RISC, or nucleic acid components such as miRNAs or siRNAs. A RNAi inhibitor can be an siNA molecule, an antisense molecule, an aptamer, or a small molecule that interacts with or interferes with the function of RISC, a miRNA, or an siRNA or any other component of the RNAi pathway in a cell or organism. By inhibiting RNAi (e.g., RNAi mediated cleavage of a target polynucleotide, translational inhibition, or transcriptional silencing), a RNAi inhibitor of the invention can be used to modulate (e.g., up-regulate or down regulate) the expression of a target gene.

[0104] The phrase "sense region" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to a nucleotide sequence of an siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of an siNA molecule can comprise a nucleic acid sequence having homology or sequence identity with a target nucleic acid sequence. In one embodiment, the sense region of the siNA molecule is also referred to as the sense strand or passenger strand.

[0105] The phrases "short interfering nucleic acid", "siNA", "short interfering RNA", "siRNA", "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule", or "chemically modified short interfering nucleic acid molecule" refer to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication by mediating RNA interference ("RNAi") or gene silencing in a sequence-specific manner. These terms can refer to both individual nucleic acid molecules, a plurality of such nucleic acid molecules, or pools of such nucleic acid molecules. The siNA can be a double-stranded nucleic acid molecule comprising self-complementary sense and antisense strands, wherein the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single-stranded polynucleotide having a nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single-stranded polynucleotide can further comprise a terminal phosphate group, such as a 5' -phosphate (see for example, Martinez et al, 2002, Cell, 1 10, 563-574 and Schwarz et al , 2002, Molecular Cell, 10, 537-568), or 5\3'- diphosphate.

[01061 The term "subject" as used herein refers to its meaning as is generally accepted in the art. The term generally refers an organism to which the nucleic acid molecules of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells. The term also refers to an organism, which is a donor or recipient of explanted cells or the cells themselves.

[0107] The phrase "systemic administration" as used herein refers to its meaning as is generally accepted in the art. The term generally refers in vivo systemic absoiption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.

[0108] The term "target" as it refers to MAP l refers to any MAPK1 target protein, peptide, or polypeptide, such as encoded by Genbank Accession Nos. shown in Table 7. The term also refers to nucleic acid sequences or target polynucleotide sequence encoding any target protein, peptide, or polypeptide, such as proteins, peptides, or polypeptides encoded by sequences having Genbank Accession Nos. shown in Table 7. The target of interest can include target polynucleotide sequences, such as target DNA or target KNA. The term "target" is also meant to include other sequences, such as differing isoforms, mutant target genes, splice variants of target polynucleotides, target polymorphisms, and non-coding (e.g., ncRNA, miRNA, stRNA, sRNA) or other regulatory polynucleotide sequences as described herein.

[0109] The phrase "target site" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a sequence within a target nucleic acid molecule, (e.g., RNA) that is "targeted", e.g., for cleavage mediated by an siNA construct, which contains sequences within its antisense region that are complementary to the target sequence.

[0110] The phrase "therapeutically effective amount" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to the amount of the compound or composition that will elicit the biological or medical response of a cell, tissue, system, animal or human that is be sought by the researcher, veterinarian, medical doctor or other clinician. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is that amount necessary to effect at least a 25% reduction in that parameter.

[0111] The phrase "universal base" as used herein refers to its meaning as is generally accepted in the art. The term universal base generally refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little or no discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3- nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example, Loakes, 2001 , Nucleic Acids Research, 29, 2437-2447).

[0112] The term "up-regulate" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to an increase in the expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more RNAs, proteins or protein subunits, above that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In certain instances, up-regulation or promotion of gene expression with an siNA molecule is above that level observed in the presence of an inactive or attenuated molecule. In other instances, up-regulation or promotion of gene expression with siNA molecules is above that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In still other instances, up-regulation or promotion of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence. In some instances, up-regulation or promotion of gene expression is associated with inhibition of RNA mediated gene silencing, such as RNAi mediated cleavage or silencing of a coding or non-coding RNA target that down regulates, inhibits, or silences the expression of the gene of interest to be up-regulated. The down regulation of gene expression can, for example, be induced by a coding RNA or its encoded protein, such as through negative feedback or antagonistic effects. The down regulation of gene expression can, for example, be induced by a non-coding RNA having regulatory control over a gene of interest, for example by silencing expression of the gene via translational inhibition, chromatin structure, methylation, RISC mediated RNA cleavage, or translational inhibition. As such, inhibition or down regulation of targets that down regulate, suppress, or silence a gene of interest can be used to up- regulate expression of the gene of interest toward therapeutic use.

[0113] The term "vector" as used herein refers to its meaning as is generally accepted in the art. The term vector generally refers to any nucleic acid- and/or viral-based expression system or technique used to deliver one or more nucleic acid molecules.

B. siNA Molecules of the Invention

[0114] The present invention provides compositions and methods comprising siNAs targeted to MAPK1 that can be used to treat diseases, e.g., malignancies and/or cancers associated with MAP 1 expression. In particular aspects and embodiments of the invention, the nucleic acid molecules of the invention comprise at least a 15 nucleotide sequence of the sequences shown in Table la and Table lb. The siNAs can be provided in several forms. For example, the siNA can be isolated as one or more siNA compounds, or it may be in the form of a transcriptional cassette in a DNA plasmid. The siNA may also be chemically synthesized and can include modifications as shown, for example, but not limitation, in Table lc and Table 8. Thus, in various embodiments, at least one strand or region of the nucleic acids of the invention comprises at least a 15 nucleotide sequence selected from the group of sequences consisting of SEQ ID NOS:l-1462. The siNAs can be administered alone or co-administered with other siNA molecules or with conventional agents that treat a MAPK1 related disease or condition.

[0115] The siNA molecules of the invention can be used to mediate gene silencing, specifically MAPK1 , via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in modulation of gene silencing either at the transcriptional level or post-transcriptional level such as, for example, but not limited to, RNAi or through cellular processes that modulate the chromatin structure or methylation patterns of the target and prevent transcription of the target gene, with the nucleotide sequence of the target thereby mediating silencing. More specifically, the target is any of MAPK1 RNA, DNA, or mRNA,

[0116] In one aspect, the invention provides short interfering nucleic acid (siNA) molecules for inhibiting the expression of the MAPK1 gene in a cell or mammal. The siNA can be single- stranded or double-stranded. When double-stranded, the siNA comprising a sense and an antisense stand. The antisense strand is complementary to at least a part of an mRNA formed in the expression of the MAPKl gene. The sense strand comprises a region that is complementary to the antisense strand. In specific embodiments, the antisense strand comprises at least a 15 nucleotide sequence of an antisense sequence listed in Table lb. Generally, the double-stranded siNA comprises at least a 15 nucleotide sequence of the sense strand in Table lb and at least a 15 nucleotide sequence of the antisense strand in Table lb. One or more of the nucleotides of the siNAs of the invention are optionally modified. In further embodiments having modifications, some siNAs of the invention comprises at least one nucleotide sequence selected from the groups of sequences provide in Table lc. In other embodiments, the siNA comprises at least two sequences selected from the group of sequences provided in Table lc, wherein one of the at least two sequences is complementary to another of the at least two sequences and one of the at least two sequences is complementary to a sequence of a mRNA generated in the expression of the MAPKl gene. Examples of certain modified siNAs of the invention are in Table lc.

[0117] The double stranded RNA molecules of the invention can comprise two distinct and separate strands that can be symmetric or asymmetric and are complementary, i.e., two single- stranded RNA molecules, or can comprise one single-stranded molecule in which two complementary portions, e.g., a sense region and an antisense region, are base-paired, and are covalently linked by one or more single-stranded "hairpin" areas (i.e. loops) resulting in, for example, a single-stranded short-hairpin polynucleotide or a circular single-stranded polynucleotide.

[0118] The linker can be polynucleotide linker or a non-nucleotide linker. In some embodiments, the linker is a non-nucleotide linker. In some embodiments, a hairpin or circular siNA molecule of the invention contains one or more loop motifs, wherein at least one of the loop portions of the siNA molecule is biodegradable. For example, a single-stranded hairpin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising 1 , 2, 3 or 4 nucleotides. Or alternatively, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising about 2 nucleotides.

[0119] In symmetric siNA molecules of the invention, each strand, the sense (passenger) strand and antisense (guide) strand, are independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. Generally, each strand of the symmetric siNA molecules of the invention are about 19-24 (e.g., about 19, 20, 21 , 22, 23 or 24) nucleotides in length.

[0120] In asymmetric siNA molecules, the antisense region or strand of the molecule is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the sense region is about 3 to about 25 (e.g. , about 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) nucleotides in length. Generally, each strand of the asymmetric siNA molecules of the invention is about 19-24 (e.g., about 19, 20, 21, 22, 23 or 24) nucleotides in length.

[0121] In yet other embodiments, siNA molecules of the invention comprise single stranded hairpin siNA molecules, wherein the siNA molecules are about 25 to about 70 (e.g. , about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length. [0122] In still other embodiments, siNA molecules of the invention comprise single-stranded circular siNA molecules, wherein the siNA molecules are about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length.

[0123] In still other embodiments, siNA molecules of the invention comprise single-stranded non-circular siNA molecules, wherein the siNA molecules are independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length.

[0124] In various symmetric embodiments, the siNA duplexes of the invention independently comprise about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs. Generally, the duplex structure of siNAs of the invention is between 15 and 30, more generally between 3 8 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length.

[0125] In yet other embodiments, where the duplex siNA molecules of the invention are asymmetric, the siNA molecules comprise about 3 to 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25) base pairs. Generally, the duplex structure of siNAs of the invention is between 15 and 25, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length.

[0126] In still other embodiments, where the siNA molecules of the invention are hairpin or circular structures, the siNA molecules comprise about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs.

[0127] The sense strand and antisense strand, or the sense region and antisense region, of the siNA molecules of the invention can be complementary. Also, the antisense strand or antisense region can be complementary to a nucleotide sequence or a portion thereof of the MAPKl target RNA. The sense strand or sense region of the siNA can comprise a nucleotide sequence of a MAPKl gene or a portion thereof. In certain embodiments, the sense region or sense strand of an siNA molecule of the invention is complementary to that portion of the antisense region or antisense strand of the siNA molecule that is complementary to a MAPKl target polynucleotide sequence, such as for example, but not limited to, those sequences represented by GENBANK Accession Nos. shown in Table 7.

[0128] In some embodiments, siNA molecules of the invention have perfect complementarity between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule. In other or the same embodiments, the antisense strand of the siNA molecules of the invention are perfectly complementary to a corresponding target nucleic acid molecule.

[0129] In yet other embodiments, siNA molecules of the invention have partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule or between the antisense strand or antisense region of the siNA molecule and a corresponding target nucleic acid molecule. Thus, in some embodiments, the double-stranded nucleic acid molecules of the invention, have between about 15 to about 30 (e.g., about 15, 16, 17, 1 8, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in one strand that are complementary to the nucleotides of the other strand. In other embodiments, the molecules have between about 15 to about 30 (e.g. , about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in the sense region that are complementaiy to the nucleotides of the antisense region, of the double-stranded nucleic acid molecule. In certain embodiments, the double-stranded nucleic acid molecules of the invention have between about 15 to about 30 (e.g. , about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in the antisense strand that are complementary to a nucleotide sequence of its corresponding target nucleic acid molecule.

[0130] In other embodiments, the siNA molecule can contain one or more nucleotide deletions, substitutions, mismatches and/or additions; provided, however, that the siNA molecule maintains its activity, for example, to mediate RNAi. In a non-limiting example, the deletion, substitution, mismatch and/or addition can result in a loop or bulge, or alternately a wobble or other alternative (iron Watson-Crick) base pair. Thus, in some embodiments, for example, the double-stranded nucleic acid molecules of the invention, have 1 or more (e.g., 1 , 2, 3, 4, 5, or 6) nucleotides, in one strand or region that are mismatches or non-base-paired with the other strand or region.. In other embodiments, the double-stranded nucleic acid molecules of the invention, have 1 or more (e.g., 1 , 2, 3, 4, 5, or 6) nucleotides in each strand or region that are mismatches or non-base-paired with the other strand or region. In a preferred embodiment, the siNA of the invention contains no more than 3 mismatches. If the antisense strand of the siNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity.

[0131] In other embodiments, the siNA molecule can contain one or more nucleotide deletions, substitutions, mismatches and/or additions to a sequence in Table lb provided, however, that the siNA molecule maintains its activity, for example, to mediate RNAi. In a non- limiting example, the deletion, substitution, mismatch and/or addition can result in a loop or bulge, or alternately a wobble or other alternative (non Watson-Crick) base pair.

[0132] The invention also comprises double-stranded nucleic acid (siNA) molecules as otherwise described hereinabove in which the first strand and second strand are complementary to each other and wherein at least one strand is hybridisable to the polynucleotide sequence of a sequence in Table lb under conditions of high stringency, and wherein any of the nucleotides is unmodified or chemically modified.

[0133] Hybridization techniques are well known to the skilled artisan (see for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm D A; followed by washing the filters in 0.1 x SSC at about 65°C.

[0134] In one specific embodiment, the first strand has about 15, 16, 17, 18, 19, 20 or 21 nucleotides that are complementary to the nucleotides of the other strand and at least one strand is hybridisable to a polynucleotide sequence in Table lb. In a more preferred embodiment, the first strand has about 15, 16, 17, 18, 19, 20 or 21 nucleotides that are complementary to the nucleotides of the other strand and at least one strand is hybridisable to SEQ ID NO: 1 , SEQ ID NO: 121 0, SEQ ID NO: 2, SEQ ID NO: 121 1 , SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO: 1213; under conditions of high stringency, and wherein any of the nucleotides is unmodified or chemically modified.

[0135] In certain embodiments, the siNA molecules of the invention comprise overhangs of about 1 to about 4 (e.g., about 1, 2, 3 or 4) nucleotides. The nucleotides in the overhangs can be the same or different nucleotides. In some embodiments, the overhangs occur at the 3'-end at one or both strands of the double-stranded nucleic acid molecule. For example, a double- stranded nucleic acid molecule of the invention can comprise a nucleotide or non-nucleotide overhang at the 3 '-end of the antisense strand/region, the 3'-end of the sense strand/region, or both the antisense strand/region and the sense strand/region of the double-stranded nucleic acid molecule.

[0136] In some embodiments, the nucleotides comprising the overhang portion of an siNA molecule of the invention comprise sequences based on the MAPK1 target polynucleotide sequence in -which nucleotides comprising the overhang portion of the antisense strand/region of an siNA molecule of the invention can be complementary to nucleotides in the MAPK1 target polynucleotide sequence and/or nucleotides comprising the overhang portion of the sense strand/region of an siNA molecule of the invention can comprise the nucleotides in the MAPK1 target polynucleotide sequence. Thus, in some embodiments, the overhang comprises a two nucleotide overhang that is complementary to a portion of the MAPK1 target polynucleotide sequence. In other embodiments, however, the overhang comprises a two nucleotide overhang that is not complementary to a portion of the MAPK1 target polynucleotide sequence. In certain embodiments, the overhang comprises a 3'-UU overhang that is not complementary to a portion of the MAP 1 target polynucleotide sequence. In other embodiments, the overhang comprises a UU overhang at the 3' end of the antisense strand and a TT overhang at the 3' end of the sense strand. In other embodiments, the overhang comprises nucleotides as described in the examples, Tables, and Figures herein.

[0137] In any of the embodiments of the siNA molecules described herein having 3'-terminaI nucleotide overhangs, the overhangs are optionally chemically modified at one or more nucleic acid sugar, base, or backbone positions. Representative, but not limiting examples of modified nucleotides in the overhang portion of a double-stranded nucleic acid (siNA) molecule of the invention include: 2'-0-alkyl (e.g., 2'-0-methyl), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-deoxy-2'- fluoroarabino (FANA), 4'-thio, 2'-0-trifluoromethyl, 2'-0-ethyl-trifluoromethoxy, 2'-0- difluoromethoxy-ethoxy, universal base, acyclic, or 5-C-methyl nucleotides. In more preferred embodiments, the overhang nucleotides are each independently, a 2'-0-alkyl nucleotide, a 2 -0- methyl nucleotide, a 2'-dexoy-2-fluoro nucleotide, or a 2'-deoxy ribonucleotide. In some instances the overhang nucleotides are linked by a one or more phosphorothioate linkages.

[0138] In yet other embodiments, siNA molecules of the invention comprise duplex nucleic acid molecules with blunt ends (i.e., without nucleotide overhangs), where both ends are blunt, or alternatively, where one of the ends is blunt. In some embodiments, the siNA molecules of the invention can comprises one blunt end, for example wherein the 5'-end of the antisense strand and the 3 '-end of the sense strand do not have any overhanging nucleotides. In another example, the siNA molecule comprises one blunt end, for example wherein the 3 '-end of the antisense strand and the 5 '-end of the sense strand do not have any overhanging nucleotides. In other embodiments, siNA molecules of the invention comprise two blunt ends, for example wherein the 3 '-end of the antisense strand and the 5 '-end of the sense strand as well as the 5 '-end of the antisense strand and 3'-end of the sense strand do not have any overhanging nucleotides.

[0139] In any of the embodiments or aspects of the siNA molecules of the invention, the sense strand and/or the antisense strand can further have a cap, such as described herein or as known in the art, at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand and/or antisense strand. Or as in the case of a haiipin siNA molecule, the cap can be at either one or both of the terminal nucleotides of the polynucleotide. In some embodiments, the cap is at one of both of the ends of the sense strand of a double-stranded siNA molecule. In other embodiments, the cap is at the 3'-end of antisense (guide) strand. In preferred embodiments, the caps are at the 3'-end of the sense strand and the 5'-end of the sense strand.

[0140] Representative, but non-limiting examples of such terminal caps include an inverted abasic nucleotide, an inverted deoxy abasic nucleotide, an inverted nucleotide moiety, a group shown in Figure 5, a glyceryl modification, an alky! or cycloalkyl group, a heterocycle, or any other cap as is generally known in the art. [0141] Any of the embodiments of the siNA molecules of the invention can have a 5' phosphate termini. In some embodiments, the siNA molecules lack terminal phosphates.

[0142] Any siNA molecule or construct of the invention can comprise one or more chemical modifications. Modifications can be used to improve in vitro or in vivo characteristics such as stability, activity, toxicity, immune response (e.g., prevent stimulation of an interferon response, an inflammatory or pro-inflammatory cytokine response, or a Toll-like Receptor (TIF) response), and/or bioavailability.

[0143] Applicants describe herein chemically modified siNA molecules with improved RNAi activity and/or stability compared to corresponding unmodified siNA molecules. Various chemically modified siNA motifs disclosed herein provide the capacity to maintain RNAi activit that is substantially similar to unmodified or minimally modified active siR A (see for example Elbashir et al., 2001 , EMBO J., 20:6877-6888) while at the same time providing nuclease resistance and pharmacokinetic properties suitable for use in therapeutic applications.

[0144] In various embodiments, the siNA molecules of the invention comprise modifications wherein any (e.g., one or more or all) nucleotides present in the sense and/or antisense strand are modified nucleotides (e.g., wherein one nucleotide is modified, some nucleotides (i.e., plurality or more than one) are modified, or all nucleotides are modified nucleotides. In some embodiments, the siNA molecules of the invention are partially modified (e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 1 7, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, or 59 nucleotides are modified) with chemical modifications. In some embodiments, an siNA molecule of the invention comprises at least about 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60 nucleotides that are modified nucleotides. In other embodiments, the siNA molecules of the invention are completely modified (e.g., 100% modified) with chemical modifications, i.e., the siNA molecule does not contain any ribonucleotides. In some of embodiments, one or more of the nucleotides in the sense strand of the siNA molecules of the invention are modified. In the same or other embodiments, one or more of the nucleotides in the antisense strand of the siNA molecules of the invention are modified. [0145] The chemical modification within a single siNA molecule can be the same or different. In some embodiments, at least one strand has at least one chemical modification. In other embodiments, each strand has at least one chemical modifications, which can be the same or different, such as, sugar, base, or backbone (i.e., internucleotide linkage) modifications. In other embodiments, siNA molecules of the invention contain at least 2, 3, 4, 5, or more different chemical modifications.

[0146] Non-limiting examples of chemical modifications that are suitable for use in the present invention, are disclosed in U.S. Patent Applications Nos. 10/444,853; 10/981 ,966; 12/064,014 and in references cited therein and include sugar, base, and phosphate, non- nucleotide modifications, and/or any combination thereof.

[0147] In certain specific embodiments of the invention, at least one modified nucleotide is a 2'-deoxy-2-fluoro nucleotide, a 2'-deoxy nucleotide, a 2'-0-alkyl (e.g., 2'-0-methyl) nucleotide, or a locked nucleic acid (LNA) nucleotide as is generally recognized in the art.

[0148] In yet other embodiment of the invention, at least one nucleotide has a ribo-like, Northern or A form helix configuration (see e.g., Saenger, Principles of Nucleic Acid Structure, Springer- Verlag ed., 1984). Non-limiting examples of nucleotides having a Northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-0, 4^'-C-methylene-(D- ribofuranosyl) nucleotides); 2^!-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl nucleotides, 2'-deoxy-2'-fluoro nucleotides; 2'-deoxy-2'-chloro nucleotides; 2'-azido nucleotides; 2'-0-trifluoromethyl nucleotides; 2'-0-ethyl-trifluoromethoxy nucleotides; 2'-0- difluoromethoxy-ethoxy nucleotides; 4'-thio nucleotides and 2'-0-methyl nucleotides.

[0149] In various embodiments, a majority (e.g., greater than 50%) of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In some of the same and/or other embodiments, a majority (e.g., greater than 50%) of the purine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

[0150] In some embodiments, the pyrimidine nucleotides in the antisense strand are 2'-0- methyl or 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense strand are 2'-0-methyl nucleotides or 2'-deoxy nucleotides. In other embodiments, the pyrimidine nucleotides in the sense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense strand are 2'-0-methyl or 2'-deoxy purine nucleotides.

[0151] In certain embodiments of the invention, all the pyrimidine nucleotides in the complementary region on the sense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides, in certain embodiments, all of the pyrimidine nucleotides in the complementary region of the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In certain embodiments, all the purine nucleotides in the complementary region on the sense strand are 2'-deoxy purine nucleotides. In certain embodiments, all of the purines in the complementary region on the antisense strand are 2'-0-methyl purine nucleotides. In certain embodiments, all of the pyrimidine nucleotides in the complementary regions on the sense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides; all of the pyrimidine nucleotides in the complementary region of the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides; all the purine nucleotides in the complementary region on the sense strand are 2'-deoxy purine nucleotides and all of the purines in the complementary region on the antisense strand are 2'-0-methyl purine nucleotides.

[0152] In some embodiments, at least 5 or more of the pyrimidine nucleotides in one or both stands are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In some embodiments, at least 5 or more of the pyrimidine nucleotides in one or both stands are 2'-0-methyl pyrimidine nucleotides. In some embodiments, at least 5 or more of the purine nucleotides in one or both stands are 2'- deoxy-2'-fluoro purine nucleotides In some embodiments, at least 5 or more of the purine nucleotides in one or both stands are 2'-0-methyl purine nucleotides.

[0153] In certain embodiments, the purines and pyrimidines are differentially modified at the 2'-sugar position (i.e., at least one purine has a different modification from at least one pyrimidine in the same or different strand at the 2'-sugar position). For example, in some instances, at least 5 or more of the pyrimidine nucleotides in one or both stands are 2'-deoxy-2'- fluoro pyrimidine nucleotides and at least 5 or more purine nucleotides in one or both strands are 2'-0-methyl purine nucleotides. In other instances at least 5 or more of the pyrimidine nucleotides in one or both stands are 2'-0-methyl pyrimidine nucleotides and at least 5 or more purine nucleotides in one or both strands are 2'-deoxy-2'-fluoro purine nucleotides. [0154] Further non-limiting examples of sense and antisense strands of such siNA molecules having various modifications and modifications patterns are shown in Figures 2 and 3.

[0155] Any of the above described modifications, or combinations thereof, including those in the references cited, can be applied to any of the siNA molecules of the invention.

[0156] The modified siNA molecules of the invention can comprise modifications at various locations within the siNA molecule. In some embodiments, the double-stranded siNA molecule of the invention comprises modified nucleotides at internal base paired positions within the siNA duplex. In other embodiments, a double-stranded siNA molecule of the invention comprises modified nucleotides at non-base paired or overhang regions of the siNA molecule. In yet other embodiments, a double-stranded siNA molecule of the invention comprises modified nucleotides at terminal positions of the siNA molecule. For example, such terminal regions include the 3'- posilion and/or 5'-position of the sense and/or antisense strand or region of the siNA molecule. Additionally, any of the modified siNA molecules of the invention can have a modification in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. Moreover, with regard to chemical modifications of the siNA molecules of the invention, each strand of the double-stranded siNA molecules of the invention can have one or more chemical modifications, such that each strand comprises a different pattern of chemical modifications.

[0157] In certain embodiments each strand of a double-stranded siNA molecule of the invention comprises a different pattern of chemical modifications, such as any Stab modification chemistries described herein (see Table 8) or any combination thereof, i.e., different combinations of defined Stabilzation chemistry (Stab) sense and antisense strands. Further, non- limiting examples of modification schemes that could give rise to different patterns of modifications are shown in Table 8. The stabilization chemistries referred to in Table 8 as Stab, can be combined in any combination of sense/antisense chemistries, such as Stab 7/8, Stab 7/1 1 , Stab 8/8, Stab 18/8, Stab 18/1 1, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab 8/32, or Stab 18/32 or any other combination of Stabilization chemistries. [0158] In any of the siNAs of the invention, one or more (for example 1 , 2, 3, 4 or 5) nucleotides at the 5'-end of the guide strand or guide region (also known as antisense strand or antisense region) of the siNA molecule are ribonucleotides.

[0159] In certain embodiments, the present invention provides a double-stranded short interfering nucleic acid (siNA) molecule that modulates the expression of MAPKl, wherein the siNA comprises a sense strand and an antisense strand; each strand is independently 15 to 30 nucleotides in length; and the antisense strand comprises at least 15 nucleotides having sequence complementary to any of:

5'-AGAACAUCAUUGGAAUCAA-3' (SEQ ID NO: 1);

5'-UAUCCAUUCAGCUAACGUU-3' (SEQ ID NO: 2);

5'-UCCAUUCAGCUAACGUUCU-3' (SEQ ID NO: 3); or

5^>-UUAUGAUAAUGUCAACAAA-3' (SEQ ID NO: 4).

[0160] In some embodiments, the antisense strand of a siNA molecule of the invention comprises at least a 15 nucleotide sequence of:

5'-UUGAUUCCAAUGAUGUUCU-3' (SEQ ID NO: 1210);

5'-AACGUUAGCUGAAUGGAUA-3' (SEQ ID NO: 121 1);

5'-AGAACGUUAGCUGAAUGGA-3' (SEQ ID NO: 1212); or 5'-UUUGUUGACAUUAUCAUAA-3' (SEQ ID NO: 1213).

[0161] In some embodiments, the sense strand of a siNA molecule of the invention comprises at least a 15 nucleotide sequence of:

5^>-AGAACAUCAUUGGAAUCAA-3^* (SEQ ID NO: 1 );

5'-UAUCCAUUCAGCUAACGUU-3' (SEQ ID NO: 2);

5'-UCCAUUCAGCUAACGUUCU-3' (SEQ ID NO: 3); or

5'-UUAUGAUAAUGUCAACAAA-3' (SEQ ID NO: 4).

[0162] In some embodiments, a siNA molecule of the invention comprises any of: 5 '-AGAACAUCAUUGGA AUCAA-3' (SEQ ID NO: 1) and 5'-

UUGAUUCCAAUGAUGUUCU-3' (SEQ ID NO: 1210); or

5 ' -U AUCC AUUCAGCU A ACGUU-3 (SEQ ID NO: 2); and 5'-

A ACGUU AGCUG A AUGG AU A-3 ' (SEQ ID NO: 121 1 ); or

5'-UCCAUUCAGCUAACGUUCU-3 (SEQ ID NO: 3) and 5'-

AGAACGUUAGCUGAAUGGA-3' (SEQ ID NO: 1212); or

5'-UUAUGAUAAUGUCAACAAA-3' (SEQ ID NO: 4); and 5'-

UUUGUUGACAUUAUCAUAA-3' (SEQ ID NO: 1213).

[0163] Any of the above described modifications, or combinations thereof, including those in the references cited, can be applied to any of these embodiments.

[0164] In certain embodiments, the nucleotides of the at least a 15 nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 1210, SEQ ID NO: 2, SEQ ID NO: 121 1 , SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO: 1213 form a contiguous stretch of nucleotides.

[0165] In some embodiments, the siNA molecule can contain one or more nucleotide deletions, substitutions, mismatches and/or additions to the at least 15 nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 1210, SEQ ID NO: 2, SEQ ID NO: 121 1 , SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO:1213; provided, however, that the siNA molecule maintains its activity, for example, to mediate RNAi. In a non-limiting example, the deletion, substitution, mismatch and/or addition can result in a loop or bulge, or alternately a wobble or other alternative (non Watson-Crick) base pair.

[0166] In certain embodiments of the invention, double-stranded siNA molecules are provided, wherein the molecule has a sense strand and an antisense strand and comprises the following formula (A):

B ^•N_X3

B (N)x! ^•N_X4 [Ν]χ₅ -5'

(A) wherein, the upper strand is the sense strand and the lower strand is the antisense strand of the double-stranded nucleic acid molecule; wherein the antisense strand comprises at least a 15 nucleotide sequence of SEQ ID NO: 1210, SEQ ID NO: 121 1 , SEQ ID NO: 1212, or SEQ ID NO: 1213, and the sense strand comprises a sequence having complementarity to the antisense strand; each N is independently a nucleotide which is unmodified or chemically modified or a non-nucleotide; each B is a terminal cap that is present or absent;

[N] represents nucleotides that are ribonucleotides; X 1 and X2 are independently integers from 0 to 4; X3 is an integer from 15 to 30; X4 is an integer from 9 to 30; and

X5 is an integer from 0 to 6, provided that the sum of X4 and X5 is 15-30.

[0167] In certain embodiments, the nucleotides of the at least a 15 nucleotide sequence of SEQ ID NO: 1210, SEQ ID NO: 121 1 , SEQ ID NO: 1212, or SEQ ID NO: 1213 form a contiguous stretch of nucleotides.

[0168] In some embodiments, the siNA molecule of formula A can contain one or more nucleotide deletions, substitutions, mismatches and/or additions to the at least 15 nucleotide sequence of SEQ ID NO: 1210, SEQ ID NO: 121 1 , SEQ ID NO: 1212, or SEQ ID NO: 1213; provided, however, that the siNA molecule maintains its activity, for example, to mediate RNAi. In a non-limiting example, the deletion, substitution, mismatch and/or addition can result in a loop or bulge, or alternately a wobble or other alternative (non Watson-Crick) base pair.

[0169] In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) of formula (A); wherein (a) one or more pyrimidine nucleotides in Νχ₄ positions are independently 2'-deoxy- 2'-fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof;

(b) one or more purine nucleotides in Νχ₄ positions are independently 2'-deoxy-2'- fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof;

(c) one or more pyrimidine nucleotides in Νχ₃ positions are independently 2^!-deoxy- 2'-fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof; and

(d) one or more purine nucleotides in Νχ₃ positions are independently 2'-deoxy-2'- fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof.

[0170] In certain embodiments, the invention features a double-stranded short interfering nucleic acid (siNA) molecule of formula (A); wherein

(a) 1 , 2, 3, 4, 5 or more pyrimidine nucleotides in Νχ₄ positions are 2'-deoxy-2'- fiuoro nucleotides;

(b) 1 , 2, 3, 4, 5 or more purine nucleotides in N_X4 positions are 2'-0-alkyl nucleotides;

(c) 1 , 2, 3, 4, 5 or more pyrimidine nucleotides in N_X3 positions are 2'-deoxy-2'- fluoro nucleotides; and

(d) 1 , 2, 3, 4, 5 or more purine nucleotides in N_X3 positions are 2'-deoxy nucleotides.

[0171] In certain embodiments, the invention features a double-stranded short interfering nucleic acid (siNA) molecule of formula (A); wherein

(a) 1 , 2, 3, 4, 5 or more pyrimidine nucleotides in N_X4 positions are 2'-Oalkyl nucleotides;

(b) 1 , 2, 3, 4, 5 or more purine nucleotides in Νχ₄ positions are ribonucleotides;

(c) 1 , 2, 3, 4, 5 or more pyrimidine nucleotides in Νχ₃ positions are 2'-0-alkyl nucleotides; and (d) 1 , 2, 3, 4, 5 or more purine nucleotides in Νχ₃ positions are ribonucleotides.

[0172] In certain embodiments, the invention features a double-stranded short interfering nucleic acid (siNA) molecule of formula (A); wherein

(a) 1, 2, 3, 4, 5 or more pyrimidine nucleotides in Νχ₄ positions are 2'-deoxy-2'- fluoro nucleotides;

(b) 1 , 2, 3, 4, 5 or more purine nucleotides in Νχ₄ positions are 2'-0-alkyl nucleotides;

(c) 1 , 2, 3, 4, 5 or more pyrimidine nucleotides in N_X3 positions are 2'-0-alkyl nucleotides; and

(d) 1, 2, 3, 4, 5 or more purine nucleotides in Νχ₃ positions are 2'-deoxy-2'-fluoro nucleotides.

[0173] In certain embodiments, the invention features a double-stranded short interfering nucleic acid (siNA) molecule of formula (A) further comprising one or more phosphorothioate internucleotide linkages.

[0174] In some embodiments, siNA molecules having formula A comprise a terminal phosphate group at the 5 '-end of the antisense strand or antisense region of the nucleic acid molecule.

[0175] In various embodiments, siNA molecules having formula A comprise X5 = 0, 1 , 2, or 3; each XI and X2 - 1 or 2; X3 = 18, 19, 20, 21 , 22, or 23, and X4 = 17, 18, 19, 20, 21 , 22, or 23.

[0176] In certain embodiments, siNA molecules having formula A comprise X5 = 3. In other embodiments siNA molecules having formula A comprise X5 = 0.

[0177] In certain embodiments, siNA molecules having formula A comprise XI = 2 and X2 = 2.

[0178] In various embodiments, siNA molecules having formula A comprise X5 = 0, XI = 2, and X2 = 2. In other embodiments, siNA molecules having formula A comprise X5 = 3, X I = 2, and X2 = 2. [0179] In one specific embodiment, an siNA molecule having formula A comprises X5 = 3; each XI and X2 = 2; X3 = 19, and X4 = 16..

[0180] In another specific embodiment, an siNA molecule having fonnula A comprises X5 = 0; each XI and X2 = 2; X3 = 19, and X4 = 19.

[0181] In certain embodiments, siNA molecules having formula A comprise caps (B) at the 3' and 5' ends of the sense strand or sense region.

[0182] In certain embodiments, siNA molecules having formula A comprise caps (B) at the 3'-end of the antisense strand or antisense region.

[0183] In various embodiments, siNA molecules having formula A comprise caps (B) at the 3' and 5^* ends of the sense strand or sense region and caps (B) at the 3'-end of the antisense strand or antisense region.

[0184] In yet other embodiments, siNA molecules having formula A comprise caps (B) only at the 5'-end of the sense (upper) strand of the double-stranded nucleic acid molecule.

[0185] In some embodiments, siNA molecules having formula A further comprise one or more phosphorothioate intemucleotide linkages between the nucleotides. In certain embodiments, siNA molecules having formula A comprise one or more phosphorothioate intemucleotide linkages between the first terminal (N) and the adjacent nucleotide on the 3'end of the sense strand, antisense strand, or both sense strand and antisense strands of the nucleic acid molecule. For example, a double-stranded nucleic acid molecule can comprise X I and/or X2 = 2 having overhanging nucleotide positions with a phosphorothioate intemucleotide linkage, e.g. , (NsN) where "s" indicates phosphorothioate.

[0186] In some embodiments, one or more of the nucleotides of siNA molecules having formula A have a universal base.

[0187] In certain embodiments, siNA molecules having formula A have at position 14 from the 5'-end of the antisense strand a ribonucleotide when the nucleotide at that position 14 is a purine. In other embodiments, siNA molecules having formula A have at position 14 from the 5'-end of the antisense strand a ribonucleotide, a 2'-deoxy-2'-fluoro nucleotide or a 2'-0-methyl nucleotide when the nucleotide at that position 14 is a pyrimidine nucleotide.

[0188] In some embodiments, siNA molecules having formula A comprises (N) nucleotides in the antisense strand (lower strand) that are complementary to nucleotides in a MAPK1 target polynucleotide sequence, which also has complementarity to the N and [N] nucleotides of the antisense (lower) strand.

[0189] Any of the above described modifications, or combinations thereof, discussed above as applicable to siNAs of the invention, including those in the references cited, can be applied to any of the embodiments to siNA molecules having formula A.

C. Generation/Synthesis of siNA Molecules

[0190] The siNAs of the invention can be obtained using a number of techniques known to those of skill in the art. For example the siNA can be chemically synthesized or may be encoded by plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops.). siNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) by the E coli RNase II or Dicer. These enzymes process the dsRNA into biologically active siNA (see, e.g., Yang et al., PNAS USA 99:9942-9947 (2002); Calegari et al. PNAS USA 99: 14236 (2002) Byron et al. Ambion Tech Notes; 10 (l):4-6 (2009); awaski et al., Nucleic Acids Res., 31 :981-987 (2003), Knight and Bass, Science, 293:2269- 2271 (2001) and Roberston et al., J. Biol. Chem 243 :82(1969).

1. Chemical Synthesis

[0191] Preferably, siNA of the invention are chemically synthesized. Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al, 1992, Methods in Enzy ology 21 1 , 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61 , 33-45, and Brennan, U.S. Pat. No. 6,001,31 1. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. [0192] siNA molecules without modifications are synthesized using procedures as described in Usman et al, 1987, J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990, Nucleic Acids Res., 18, 5433. These syntheses makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end that can be used for certain siNA molecules of the invention.

[0193] In certain embodiments, the siNA molecules of the invention are synthesized, deprotected, and analyzed according to methods described in U.S. Patent Nos. 6,995,259, 6,686,463, 6,673,918, 6,649,751 , 6,989,442, and U.S. Patent Application No. 10/190,359.

[0194] In a non-limiting synthesis example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μηιοΐ scale protocol with a 2.5 min coupling step for 2'-0-methylated nucleotides and a 45 second coupling step for 2'-deoxy nucleotides or 2'-deoxy-2'-fluoro nucleotides. Table 9 outlines the amounts and the contact times of the reagents used in the synthesis cycle.

[0195] Alternatively, the siNA molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al, 1992, Science 256, 9923; Draper et al, International PCT Publication No. WO 93/23569; Shabarova et al, 1991 , Nucleic Acids Research 19, 4247; Bellon et al, 1997, Nucleosides & Nucleotides, 16, 951 ; Bellon et al, 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

[0196] Various siNA molecules of the invention can also be synthesized using the teachings of Scaringe et al, US Patent Nos. 5,889,136; 6,008,400; and 6,1 1 1 ,086.

2. Vector Expression

[0197] Alternatively, siNA molecules of the invention that interact with and down-regulate gene encoding target MAPKl molecules can be expressed and delivered from transcription units (see for example Couture et al, 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. [0198] In some embodiments, pol III based constructs are used to express nucleic acid molecules of the invention. Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III), (see for example, Thompson, U.S. Patent. Nos. 5,902,880 and 6,146,886). (See also, Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al, 1991 , Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet <?i al, 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al, 1992, J. Virol., 66, 1432-41 ; Weerasinghe et al, 1991 , J. Virol, 65, 5531-4; Ojwang et al, 1992, Proc, Natl Acad. Sci. USA, 89, 10802-6; Chen et al, 1992, Nucleic Acids Res., 20, 4581 -9; Sarver et al, 1990 Science, 247, 1222-1225; Thompson et al, 1995, Nucleic Acids Res., 23, 2259; Good et al, 1997, Gene Therapy, 4, 45. Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci, U S A, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al, 1993, Methods Enzymol, 217, 47-66; Zhou et al, 1990, Mol. Cell Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al, 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al, 1992, Proc. Natl Acad. Sci. U SA, 89, 10802-6; Chen et al, 1992, Nucleic Acids Res., 20, 4581- 9; Yu et al, 1993, Proc. Natl. Acad. Sci, U S A, 90, 6340-4; L'Huillier et al. 1992, EMBO J., 1 1 , 441 1-8; Lisziewicz et al, 1993, Proc. Natl Acad. Sci, U A, 90, 8000-4; Thompson et al, 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al, supra; Couture and Stinchcomb, 1996, supra; Noonberg et al, 1994, Nucleic Acid Res., 22, 2830; Noonberg et al, U.S. Pat. No. 5,624,803; Good et al, 1997, Gene Ther., 4, 45; Beigelman et al, International PCT Publication No. WO 96/18736. The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors). or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

[0199] Vectors used to express the siNA molecules of the invention can encode one or both strands of an siNA duplex, or a single self-complementary strand that self hybridizes into an siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al, 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al, 2002, Nature Biotechnology, 19, 500; and Novina et al, 2002, Nature Medicine, advance online publication doi: 10.1038/nm725).

D. Carrier Delivery Systems

[0200] The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or as a recombinant plasmid or viral vectors which express the siNA molecules, or otherwise delivered to target cells or tissues. Methods for the deliveiy of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; Deliveiy Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al, 1999, Mol. Membr. Biol, 16, 129- 140; Hofland and Huang, 1999, Handb. Exp. Pharmacol, 137, 165-192; and Lee et al, 2000, ACS Symp. Ser., 752, 184- 192. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al, PCT WO 94/02595 further describe the general methods for delivei of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example, Gonzalez et al, 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al. International PCT Publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example US Patent 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). [0201J In one aspect, the present invention provides carrier systems containing the siNA molecules described herein. Tn some embodiments, the carrier system is a lipid-based carrier system, cationic lipid, or liposome nucleic acid complexes, a liposome, a micelle, a virosome, a lipid nanoparticle or a mixture thereof. In other embodiments, the carrier system is a polymer- based carrier system such as a cationic polymer-nucleic acid complex. In additional embodiments, the carrier system is a cyclodextrin-based carrier system such as a cyclodextrin polymer-nucleic acid complex. In further embodiments, the carrier system is a protein-based carrier system such as a cationic peptide-nucleic acid complex. Preferably, the carrier system is a lipid nanoparticle ("LNP") formulation.

[0202] In certain embodiments, the siNA molecules of the invention are formulated with a lipid nanoparticle composition such as is described in U.S. Patent Application Nos. 1 3/353,630, 1 1 /586,102, 61/189,295, 61/204,878, 61/235,476, 61/249,807, 61/298,022, 61/351373, 61/347640, 61/345754, 61/322054, 12/640342, and 12/617079, and PCT Applications Nos. PCT/USlO/020013 and PCT/US09/ 053336. In certain preferred embodiments, the siNA molecules of the invention are formulated with a lipid nanoparticle composition comprising a cationic lipid/Cholesterol/PEG-C-DMA/DSPC in a 40/48/2/10 ratio or a cationic lipid/Cholesterol PEG-DMG/DSPC in a 40/48/2/10 ratio. In more certain embodiments, the cationic lipid is DLinDM (see Table 11), the PEG is PEG-DMG, and the N/P ratio of the formulation is 2.8. In more preferred embodiments, the cationic lipid is CLinDMA (see Tables 10 & 11).

[0203] In various embodiments, lipid nanoparticle fonnulations described in Table 10 are applied to any siNA molecule or combination of siNA molecules herein. In some embodiments, the invention features a composition comprising an siNA molecule of the invention formulated as any of formulation LNP-051 ; LNP-053; LNP-054; LNP-069; LNP-073; LNP-077; LNP-080; LNP-082; LNP-083; LNP-060; LNP-061 ; LNP-086; LNP-097; LNP-098; LNP-099; LNP-100; LNP-101 ; LNP-102; LNP-103; or LNP-304 (see Table 10).

[0204] In certain other embodiments, the invention features a composition comprising an siNA molecule of the invention formulated with any of the cationic lipid fonnulations described in U.S. Patent Application Nos. 61/189,295, 61/204,878, 61/235,476, 61/249,807, and 61/298,022.

[0205] In other embodiments, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the phannacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. Non-limiting, examples of such conjugates are described in U.S. Publication Nos. US2008/0152661 Al and US 2004/0162260 Al (e.g., CDM-LBA, CDM-Pip-LBA, CDM-PEG, CDM-NAG, etc.) and U.S. Patent Application Nos. 10/427, 160 10/201 ,394, 61/322422, and 61 /315223; and U.S. Patent Nos. 6,528,631 ; 6,335,434; 6,235,886; 6,153,737; 5,214, 136; and 5,1 38,045.

[0206] In various embodiments, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 100 to about 50.000 daltons (Da).

[0207] In yet other embodiments, the invention features compositions or formulations comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) and siNA molecules of the invention, such as is disclosed in for example, International PCT Publication No. WO 96/10391 ; Ansell et al, International PCT Publication No. WO 96/10390; Holland et al, International PCT Publication No. WO 96/10392.

[0208] In some embodiments, the siNA molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acid molecules of the invention are formulated as described in U.S. Patent Application Publication No. 20030077829. [0209] In other embodiments, siNA molecules of the invention are complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666. In still other embodiments, the membrane disruptive agent or agents and the siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Patent No. 6,235,310.

[0210] In certain embodiments, siNA molecules of the invention are complexed with delivery systems as described in U.S. Patent Application Publication Nos. 2003077829; 20050287551 ; 20050164220; 20050191627; 200501 18594; 20050153919; 20050085486; and 20030158133; and International PCT Publication Nos. WO 00/03683 and WO 02/087541.

[0211] In some embodiments, a liposomal formulation of the invention comprises an siNA molecule of the invention (e.g., siNA) formulated or complexed with compounds and compositions described in U.S. Patent Nos. 6,858,224; 6,534,484; 6,287,591 ; 6,835,395; 6,586,410; 6,858,225; 6,815,432; 6,586,001 ; 6,120,798; 6,977,223; 6,998,1 15; 5,981 ,501 ; 5,976,567; 5,705,385; and U.S. Patent Application Publication Nos. 2006/0019912; 2006/0019258; 2006/0008909; 2005/0255153; 2005/0079212; 2005/0008689; 2003/0077829, 2005/0064595, 2005/0175682, 2005/01 18253; 2004/0071654; 2005/0244504; 2005/0265961 and 2003/0077829.

[0212] Alternatively, recombinant plasmids and viral vectors, as discussed above, which express siNAs of the invention can be used to deliver the molecules of the invention. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al, 1996, TIG., 12, 510). Such recombinant plasmids can also be administered directly or in conjunction with a suitable delivery reagents, including, for example, the Mirus Transit LTl lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes lipid-based carrier system, cationic lipid, or liposome nucleic acid complexes, a micelle, a virosome, a lipid nanoparticle.

E. Kits [0213] The present invention also provides nucleic acids in kit form. The kit may comprise a container. The kit typically contains a nucleic acid of the invention with instructions for its administration. In certain instances, the nucleic acids may have a targeting moiety attached. Methods of attaching targeting moieties (e.g. antibodies, proteins) are known to those of skill in the art. In certain instances, the nucleic acids are chemically modified. In other embodiments, the kit contains more than one siNA molecule of the invention. The kits may comprise an siNA molecule of the invention with a pharmaceutically acceptable carrier or diluent. The kits may further comprise excipients.

F. Therapeutic Uses/Pharmaceutical Compositions

[0214] The present body of knowledge in MAPKl research indicates the need for methods to assay MAPKl activity and for compounds that can regulate MAPKl expression for research, diagnostic, and therapeutic use. As described infra, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of MAPKl levels. In addition, the nucleic acid molecules and pharmaceutical compositions can be used to treat disease states related to MAPKl RNA levels.

1. Disease States Associated with MAPKl

[0215] Particular disease states that can be associated with MAPKl expression modulation include various cancers including solid tumors. Non-limiting examples of such cancers include: bladder cancer, bladder transitional cell carcinoma, urothelial carcinoma, brain cancer, gliomas, astrocytomas, breast cancer, breast carcinoma, cervical cancer, colorectal cancer, rectal cancer, colorectal carcinoma, colon cancer, hereditary nonpolyposis colorectal cancer, endometrial carcinoma, esophageal cancer, esophageal squamous cell carcinoma, ocular melanoma, uveal melanoma, intraocular melanoma, primary intraocular lymphoma, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, leukemia, acute lymocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid (CML), chronic myelomonocytic (CMML), liver cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Non-Hodgkin lymphoma, B-cell lymphomas, T-cell lymphomas, precursor T-lymphoblastic lymphoma/leukemia, multiple myeloma, ovarian carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, mucosal melanoma, small intestine carcinomas, stomach cancer, gastric carcinoma, testicular cancer, testicular seminomas, testicular non-seminomas, thyroid cancer, papillary thyroid carcinoma, and papillary adenocarcinomas.

[0216] It is understood that the siNA molecules of the invention can degrade the target MAPKl mRNA (and thus inhibit the diseases stated above). Inhibition of a disease can be evaluated by directly measuring the progress of the disease in a subject. It can also be inferred through observing a change or reversal in a condition associated with the disease. Additionally, the siNA molecules of the invention can be used as a prophylaxis. Thus, the use of the nucleic acid molecules and pharmaceutical compositions of the invention can be used to ameliorate, treat, prevent, and/or cure these diseases and others associated with regulation of MAPKl gene expression.

2. Pharmaceutical Compositions

[0217] The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, prophylactic, cosmetic, veterinary, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications. a. Formulations

[0218] Thus, the present invention, in one aspect, also provides for phannaceutical compositions of the siNA molecules described, i.e., compositions in a pharmaceutically acceptable carrier or diluent. These phannaceutical compositions include salts, esters, or salts of such esters, of the above compounds, e.g. , acid addition salts, for example, salts of hydrochloric, hydrobromic, hydroiodic, acetic acid, and benzene sulfonic acid. Other salts include for example, sodium, potassium, manganese, ammonium, and calcium salts. These fonnulations or compositions can comprise a phannaceutically acceptable earner or diluent as is generally known in the art.

[0219] In one embodiment, the invention features a pharmaceutical composition comprising an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 1. In another embodiment, the invention features a pharmaceutical composition comprising an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 1210. In yet another embodiment, the invention features a pharmaceutical composition comprising an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 2. In still another embodiment, the invention features a pharmaceutical composition comprising an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 121 1. In another embodiment, the invention features a pharmaceutical composition comprising an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 3. In another embodiment, the invention features a pharmaceutical composition comprising an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 1212. In another embodiment, the invention features a pharmaceutical composition comprising an siNA molecule comprising at least a 35 nucleotide sequence of SEQ ID NO: 4. In yet another embodiment, the invention features a pharmaceutical composition comprising an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 1213. In still another embodiment, the invention features a phannaceutical composition comprising an siNA molecule comprising formula (A).

[0220] The siNA molecules of the invention are preferably formulated as phannaceutical compositions prior to administering to a subject, according to techniques known in the art. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art for example as described in Remington's Pharmaceutical Science, I 7^1h ed., Mack Publishing Company, Easton. Pa. (1985).

[0221] In some embodiments, pharmaceutical compositions of the invention (e.g. siNA and/or LNP formulations thereof) further comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include preservatives, flavoring agents, stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), addition of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). In addition, antioxidants and suspending agents can be used. [0222] Non-limiting examples of various types of formulations for local administration include ointments, lotions, creams, gels, foams, preparations for delivery by transdermal patches, powders, sprays, aerosols, capsules or cartridges for use in an inhaler or insufflator or drops (for example eye or nose drops), solutions/suspensions for nebulization, suppositories, pessaries, retention enemas and chewable or suckable tablets or pellets (for example for the treatment of aphthous ulcers) or liposome or microencapsulation preparations.

[0223] Ointments, creams and gels, can, for example, can be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agent and/or solvents. Non limiting examples of such bases can thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil, or a solvent such as polyethylene glycol. Various thickening agents and gelling agents can be used depending on the nature of the base. Non-limiting examples of such agents include soft paraffin, aluminum stearate, cetostearyl alcohol, polyethylene glycols, woolfat, beeswax, carboxypolymethylene and cellulose derivatives, and/or glyceryl monostearate and/or non-ionic emulsifying agents.

[0224] In one embodiment lotions can be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents or thickening agents.

[0225] In one embodiment powders for external application can be formed with the aid of any suitable powder base, for example, talc, lactose or starch. Drops can be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents, suspending agents or preservatives.

[0226] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

[0227] Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

[0228] Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate; or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0229] Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid [0230] Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, natural ly-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

[0231] Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such fonnulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0232] The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

[0233] Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. [0234] In other embodiments, the siNA and LNP compositions and formulations provided herein for use in pulmonary delivery further comprise one or more surfactants. Suitable surfactants or surfactant components for enhancing the uptake of the compositions of the invention include synthetic and natural as well as full and truncated forms of surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant Protein E, di- saturated phosphatidylcholine (other than dipalmitoyl), dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine; phosphatidic acid, ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols, sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycero-3- phospbocholine, dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, choline phosphate; as well as natural and artificial lamellar bodies which are the natural carrier vehicles for the components of surfactant, omega-3 fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid, non-ionic block copolymers of ethylene or propylene oxides, polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomeric and polymeric, poly (vinyl amine) with dextran and/or alkanoyl side chains, Brij 35, Triton X-100 and synthetic surfactants ALEC. Exosurf, Survan and Atovaquone, among others. These surfactants can be used either as single or part of a multiple component surfactant in a formulation, or as covalently bound additions to the 5' and/or 3' ends of the nucleic acid component of a pharmaceutical composition herein. b. Combinations

[0235] The siNAs and pharmaceutical formulations according to the invention can be administered to a subject alone or used in combination with or include one or more other therapeutic agents, for example, anticancer agents. Thus, combinations of the presently disclosed compounds with other anti-cancer or chemotherapeutic agents are within the scope of the invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita and S. Hellman (editors), 6^th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Such anti-cancer agents include, but are not limited to, the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG- CoA reductase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, apoptosis inducing agents and agents that interfere with cell cycle checkpoints. The siNAs of the invention are also useful in combination with any therapeutic agent used in the treatment of HCC, for example, but not limitation sorafenib. The instant compounds are particularly useful when co-administered with radiation therapy.

[0236] In a further embodiment, therefore, the invention provides a combination comprising an siNA molecule of the invention, such as for example, but not limitation, an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 1210, SEQ ID NO: 2, SEQ ID NO: 121 1 , SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO: 1213; or formula (A) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof together with one or more anti-cancer or chemotherapeutic agents.

[0237] In certain embodiments, the instant siNA molecules of the invention are useful in combination with known anti-cancer agents including the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, and other angiogenesis inhibitors.

[0238] Examples of estrogen receptor modulators that can be used in combination with the compounds of the invention include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381 , LY1 17081 , toremifene, fulvestrant, 4-[7-(2,2-dimethyl-l -oxopropoxy-4-methyl-2- [4-[2-(l-piperidinyl)ethoxy]phenyl]-2H- l -benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4'-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

[0239] Examples of androgen receptor modulators that can be used in combination with the compounds of the invention include, but are not limited to, finasteride and other 5ct-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.

[0240] Examples of such retinoid receptor modulators that can be used in combination with the compounds of the invention include, but are not limited to, bexarotene, tretinoin, 13-cis- retinoic acid, 9-cis-retinoic acid, a-difluoromethylornithine, ILX23-7553, trans-N-(4'- hydroxyphenyl) retinamide, and N-4-carboxyphenyl retinamide.

[0241] Examples of cytotoxic agents that can be used in combination with the compounds of the invention include, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu-(hexane-l ,6-diamine)-mu- [diamine-platinum(II)]bis[diamine(chloro)platinum (II)]tetrachloride, diarizidinylspermine, arsenic trioxide, 1-( 1 1-dodecylamino-l 0-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin. amrubicin, antineoplaston, 3'-deamino-3'-moφholino-13-deoxo- 10-hydro ycarminomycin, annamycin, galarubicin, elinafide, MEN 10755, and 4-demethoxy-3-deamino-3-aziridinyl-4- methylsulphonyl-daunorubicin (see WO 00/50032).

[0242] An example of a hypoxia activatable compound that can be used in combination with the compounds of the invention is tirapazamine.

[0243] Examples of proteasome inhibitors that can be used in combination with the compounds of the invention include, but are not limited to, lactacystin and bortezomib.

[0244] Examples of microtubule inhibitors/microtubule-stabilising agents that can be used in combination with the compounds of the invention include, but are not limited to, paclitaxel, vindesine sulfate, 3\4'-didehydro-4'-deoxy-8'-norvincaleukoblastine, docetaxol, rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881 , BMS 184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-va!yl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t- butylamide, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS 188797. [0245] Some examples of topoisomerase inhibitors that can be used in combination with the compounds of the invention include, but are not limited to, are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3',4'-0-exo-benzylidene-chartreusin, 9-methoxy-N,N- dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, l-amino-9-ethyl-5-fluoro-2,3- dihydro-9-hydroxy-4-methyl-lH,12H-benzo[de]pyrano[3',4' :b,7]-indolizino[l ,2b]quinoline- 10, 13(9H, 15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethy!]-(20S)camptothecin, BNP1350, BNPI1 100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2'- dimethylamino-2'-deoxy-etoposide, GL331 , N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6- dimethyl-6H-pyrido[4,3-b]carbazole-l-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2- (dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]- 5,5a,6,8,8a,9-hexohydrofui (3',4' :6,7)naphtho(2,3-d)-l ,3-dioxol-6-one, 2,3-(methylenedioxy)-5- methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2- aminoethyl)amino]benzo[g]isoguinoline-5,10-dione, 5-(3-aminopropylamino)-7, 10-dihydroxy-2- (2-hydroxyethy3aminomethyl)-6H-pyrazolo[4,5,l -de]acridin-6-one, N-[l - [2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2- (dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy- 7H-indeno[2,l -c] quinolin-7-one, and dimesna.

[0246] Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kinesin KSP, that can be used in combination with the compounds of the invention include, but are not limited to, inhibitors described in PCT Publications WO 01/30768, WO 01 /98278, WO 03/050,064, WO 03/050, 122, WO 03/049,527, WO 03/049,679, WO 03/049,678, WO04/039774, WO03/079973, WO03/09921 1 , WO03/3 05855, WO03/106417, WO04/037171 , WO04/058148, WO04/058700, WO04/126699, WO05/018638, WO05/019206, WO05/019205, WO05/018547, WO05/017190, US2005/0176776. In an embodiment inhibitors of mitotic kinesins include, but are not limited to inliibitors of KSP, inhibitors of MKLP l , inhibitors of CENP-E, inhibitors of MCAK, inhibitors of Kifl4, inhibitors of Mphosphl and inhibitors of Rab6-KIFL.

[0247] Examples of "histone deacetylase inhibitors" that can be used in combination with the compounds of the invention include, but are not limited to, TSA, oxamflatin, PXD 101 , MG98, valproic acid and scriptaid. Further reference to other histone deacetylase inliibitors may be found in the following manuscript; Miller, T.A. et al. J. Med. Chem. 46(24):5097-51 16 (2003). [0248] Inhibitors of kinases involved in mitotic progression that can be used in combination with the compounds of the invention include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK) (in particular inhibitors of PLK-1), inhibitors of bub-1 and inhibitors of bub-Rl .

[0249] Antiproliferative agents that can be used in combination with the compounds of the invention include, but are not limited to, antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001 , and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifiuridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylidenecytidine, 2'- fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dihydro-benzofuiyl)sullbnyl]-N'-(3,4- dichIorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero- B-L-manno-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo- 4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][l ,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glu^ acid, aminopterin, 5-flurouracil, alanosine, 1 l-acetyl-8-(carbamoyloxymethyl)-4-formyl-6- methoxy-14-oxa-l ,l l-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl- l-B-D- arabino furanosyl cytosine and 3-aminopyridine-2-carboxaldehyde thiosemicarbazone.

[0250] Examples of monoclonal antibody targeted therapeutic agents that can be used in combination with the compounds of the invention include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody, such as, for example, Bexxar.

[0251] Examples of HMG-CoA reductase inhibitors that may be used that can be used in combination with the compounds of the invention include, but are not limited to, lovastatin

(MEVACOR®; see U.S. Pat. Nos. 4,231 ,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S.

Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589), f!uvastatin (LESCOL®; see U.S. Pat. Nos. 5,354,772, 4,91 1 ,165, 4,929,437, 5, 189, 164, 5, 1 18,853, 5,290,946 and

5,356,896) and atorvastatin (LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681 ,893, 5,489,691 and 5,342,952). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 85-89 (5 February 1996) and U.S. Patent Nos. 4,782,084 and 4,885,314.

[0252] Examples of prenyl-protein transferase inhibitors that can be used in combination with the compounds of the invention include, but are not limited to, can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701 , WO 97/23478, WO 97/38665, WO 98/28980, WO 98/291 19, WO 95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat. No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S. Pat. No. 5,602,098, European Patent Publ. 0 618 221 , European Patent Publ. 0 675 1 12, European Patent Publ. 0 604 181 , European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/1 1917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No. 5,661 ,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO 96/2461 1 , WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Pat. No. 5,571 ,792, WO 96/17861 , WO 96/33159, WO 96/34850, WO 96/34851 , WO 96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31 1 1 1, WO 96/31477, WO 96/31478, WO 96/31501 , WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of a prenyl-protein transferase inhibitor on angiogenesis see European . of Cancer, Vol. 35, No. 9, pp.1394- 1401 (1999).

[0253] Examples of angiogenesis inhibitors that can be used in combination with the compounds of the invention include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Fit- 1 (VEGFR1 ) and Flk-l/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-a, interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyc!ooxy-genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1 992); MCI, Vol. 69, p. 475 (1982); Arch. OpthalmoL, Vol. 108, p.573 (1990); Anat. Rec, Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol, Vol. 16, p.107 (1996); Jpn. J. Pharmacol, Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 91 16 (1999)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-0- chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1 , angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. 105: 141-145 (1985)), and antibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp.963-968 (October 1999); Kim et al., Nature, 362, 841 -844 (1 93); WO 00/44777; and WO 00/61 1 86).

[0254] Other therapeutic agents that modulate or inhibit angiogenesis may also be used in combination with the compounds of the instant invention and include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis pathways that can be used in combination with the compounds of the invention include, but are not limited to, heparin (see Thromh. Haemost. 80: 10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101 :329-354 (2001)). TAFIa inhibitors have been described in PCT Publication WO 03/013,526 and U.S. Ser. No. 60/349,925 (filed January 18, 2002).

[0255] Agents that interfere with cell cycle checkpoints that can be used in combination with the compounds of the invention include, but are not limited to, inhibitors of ATR, ATM, the Chkl and Chk2 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7- hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

[0256] Agents that interfere with receptor tyrosine kinases (RTKs) that can be used in combination with the compounds of the invention include, but are not limited to, inhibitors of c- Kit, Eph, PDGF, Flt3 and MAPK1 . Further agents include inhibitors of RTKs as described by Bume-Jensen and Hunter, Nature, 41 1 :355-365, 2001 .

[0257] Inhibitors of cell proliferation and survival signaling pathway that can be used in combination with the compounds of the invention include, but are not limited to, inhibitors of EGFR (for example gefitinib and eriotinib), inliibitors of ERB-2 (for example trastuzumab), inhibitors of IGFR, inhibitors of cytokine receptors, inhibitors of MAPK1 , inhibitors of PI3K (for example LY294002), serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140, US 2004-01 16432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041 162, WO 2004/096131 , WO 2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344), inhibitors of Raf kinase (for example BAY-43-9006), inhibitors of MEK (for example CM 040 and PD-098059) and inhibitors of mTOR (for example Wyeth CCI-779). Such agents include small molecule inhibitor compounds and antibody antagonists.

[0258] Apoptosis inducing agents that can be used in combination with the compounds of the invention include, but are not limited to, activators of TNF receptor family members (including the TRAIL receptors).

[0259] NSAIDs that are selective COX-2 inhibitors that can be used in combination with the compounds of the invention include, but are not limited to, those NSAIDs disclosed in U.S. Pat. 5,474,995, U.S. Pat. 5,861,419, U.S. Pat. 6,001,843, U.S. Pat. 6,020,343, U.S. Pat. 5,409,944, U.S. Pat. 5,436,265, U.S. Pat. 5,536,752, U.S. Pat. 5,550, 142, U.S. Pat. 5,604,260, U.S. 5,698,584, U.S. Pat. 5,710,140, WO 94/15932, U.S. Pat. 5,344,991 , U.S. Pat. 5,134, 142, U.S. Pat. 5,380,738, U.S. Pat. 5,393,790, U.S. Pat. 5,466,823, U.S. Pat. 5,633,272, and U.S. Pat. 5,932,598, all of which are hereby incorporated by reference.

[0260] Inhibitors of COX-2 that are particularly useful in combination with the compounds of the invention include: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and 5-chloro- 3-(4-methylsulfonyl)-phenyl-2-(2-methyl-5-pyridinyl)pyridine; or a pharmaceutically acceptable salt thereof.

[0261] Compounds that have been described as specific inhibitors of COX-2 and are therefore useful in the present invention include, but are not limited to: parecoxib, CELEBREX® and BEXTRA® or a pharmaceutically acceptable salt thereof. [0262] Angiogenesis inhibitors that can be used in combination with the compounds of the invention include, but are not limited to, endostatin, ukrain, ranpirnase, 1M862, 5-methoxy-4-[2- methyl-3-(3-methyl-2-butenyl)oxiranyl]-l -oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-l-[[3,5-dichloro-4-(4-chlorobenzoyl)-phenyl]methyl]- lH-l ,2,3- triazole-4-carboxamide,CM101 , squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N- methyl-4,2-pyrrole]-carbonylimino]-bis-(l ,3-naphthalene disulfonate), and 3-[(2,4- dimethylpynOl-5-yl)methylene]-2-indolinone (SU5416).

[0263] Tyrosine kinase inhibitors that can be used in combination with the compounds of the invention include, but are not limited to, N-(trifluoromethylphenyl)-5-methylisoxazol-4- carboxamide, 3-[(2,4-dimethylpyiTol-5-yl)methylidenyl)indolin-2-one, 17-(allylamino)-l 7- demethoxygeldanamycin, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4- moφholinyl)propo yl]qui azoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolinamine, BIBX1 382, 2,3,9,10,1 1 , 12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9- methyl-9,12-epoxy-lH-diindolo[l ,2,3-fg:3 2', r-kl]pyrrolo[3,4-i][l ,6]benzodiazocin-l -one, SH268, genistein, imatinib (STI571 ), CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H- pyrrolo[2,3-d]pyrimidinemethane sulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7- dimethoxyquinazoline, 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571 A, N-4-chloiOphenyl-4-(4-pyridylmethyl)-l -phthalazinamine, and EMD 121974.

[0264] Combinations with compounds other than anti-cancer compounds are also encompassed in the instant compositions and methods. For example, combinations of the instantly claimed compounds with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment of certain malignancies. PPAR-γ and PPAR-δ are the nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol. 31 :909-913 (1998); J. Biol. Chem. 274:9116-9121 (1999); Invest. Ophthalmol Vis. Sci. 41 :2309-2317 (2000)). More recently, PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice. {Arch. Ophthamol. 1 19:709-717 (2001)). Examples of PPAR-γ agonists and PPAR- γ/α agonists that can be used in combination with the compounds of the invention include, but are not limited to, thiazolidinediones (such as DRF2725, CS-01 1 , troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501 , MCC-555, GW2331 , GW409544, NN2344, KRP297, NP01 10, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7- dipropyl-3-trifluoromethyl-l ,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid (disclosed in USSN 09/782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy) phenoxy)propoxy)-2- ethylchromane-2-carboxylic acid (disclosed in USSN 60/235,708 and 60/244,697).

[0265] Another embodiment of the instant invention is the use of the presently disclosed compounds in combination with gene therapy for the treatment of cancer. For an overview of genetic strategies to treating cancer see Hall el al. {Am J Hum Genet 61 :785-789 (1997)) and Kufe el al. {Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton, 2000). Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134, for example), a uPA/uPAR antagonist ("Adenovirus-Mediated Delivery of a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice," Gene Therapy, August 5(8): 1 105-13 (1998)), and interferon gamma {J Immunol 164:217-222 (2000)).

[0266] The compounds of the instant invention may also be administered in combination with an inhibitor of inherent multidrug resistance (MDR), in particular MDR associated with high levels of expression of transporter proteins. Such MDR inhibitors include inhibitors of p- glycoprotein (P-gp), such as LY335979, XR9576, OC 144-093, R101922, VX853 and PSC833 (valspodar).

[0267] A compound of the present invention may be employed in conjunction with antiemetic agents to treat nausea or emesis, including acute, delayed, late-phase, and anticipatory emesis, which may result from the use of a compound of the present invention, alone or with radiation therapy. For the prevention or treatment of emesis, a compound of the present invention may be used in conjunction with other anti-emetic agents, especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as disclosed in U.S. Patent Nos. 2,789,1 18, 2,990,401 , 3,048,581 , 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, an antidopaminergic, such as the phenothiazines (for example prochlorperazine, fluphenazine, thioridazine and mesoridazine), metoclopramide or dronabinol. In an embodiment, an anti-emesis agent selected from a neurokinin- 1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is administered as an adjuvant for the treatment or prevention of emesis that may result upon administration of the instant compounds.

[0268] Neurokinin- 1 receptor antagonists of use in conjunction with the compounds of the present invention are fully described, for example, in U.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European Patent Publication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430 771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901 , 0 512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681 , 0 517 589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0 545 478, 0 558 156, 0 577 394, 0 585 913,0 590 152, 0 599 538, 0 610 793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0 707 006, 0 708 101 , 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776 893; PCT International Patent Publication Nos. WO 90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93/01 159, 93/01165, 93/01169, 93/01 170, 93/06099, 93/091 16, 93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21 155, 93/21181 , 93/23380, 93/24465, 94/00440, 94/01402, 94/02461 , 94/02595, 94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167, 94/10168, 94/10170, 94/1 1368, 94/13639, 94/13663, 94/14767, 94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/1 1880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674, 95/30687, 95/33744, 96/05181 , 96/05193, 96/05203, 96/06094, 96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661 , 96/29304, 96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931 , 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2 302 689. The preparation of such compounds is fully described in the aforementioned patents and publications, which are incorporated herein by reference.

[0269] In an embodiment, the neurokinin- 1 receptor antagonist for use in conjunction with the compounds of the present invention is selected from: 2-(R)-(l-(R)-(3,5-bis(trifluoromethyl)- phenyl)etho y)-3-(S)-(4-fluorop enyl)-4-(3-(5-oxo-l H,4H-L2,4-triazolo)methyl)mo hoiine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Pat. No. 5,719,147.

[0270] A compound of the instant invention may also be useful for treating or preventing cancer, including bone cancer, in combination with bisphosphonates (understood to include bisphosphonates, diphosphonates, bisphosphonic acids and diphosphonic acids). Examples of bisphosphonates include but are not limited to: etidronate (Didronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate, EB-1053, minodronate, neridronate, piridronate and tiludronate including any and all pharmaceutically acceptable salts, derivatives, hydrates and mixtures thereof.

[0271] A compound of the instant invention may also be administered with an agent useful in the treatment of anemia. Such an anemia treatment agent is, for example, a continuous eythropoiesis receptor activator (such as epoetin alfa).

[0272] A compound of the instant invention may also be administered with an agent useful in the treatment of neutropenia. Such a neutropenia treatment agent is, for example, a hematopoietic growth factor which regulates the production and function of neutrophils such as a human granulocyte colony stimulating factor, (G-CSF). Examples of a G-CSF include filgrastim and PEG-filgrastim.

[0273] A compound of the instant invention may also be administered with an immunologic- enhancing drug, such as levamisole, isoprinosine and Zadaxin.

[0274] A compound of the instant invention may also be useful for treating or preventing breast cancer in combination with aromatase inhibitors. Examples of aromatase inhibitors include but are not limited to: anastrozole, letrozole and exemestane. [0275] A compound of the instant invention may also be useful for treating or preventing cancer in combination with other siNA therapeutics.

[0276] The compounds of the instant invention may also be administered in combination with γ-secretase inhibitors and/or inhibitors of NOTCH signaling. Such inhibitors include compounds described in WO 01/90084, WO 02/30912, WO 01 /70677, WO 03/013506, WO 02/36555, WO 03/093252, WO 03/093264, WO 03/093251 , WO 03/093253, WO 2004/039800, WO 2004/039370, WO 2005/030731, WO 2005/014553, USSN 10/957,251, WO 2004/08991 1 , WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031 137, WO 2004/031 139, WO 2004/031 138, WO 2004/101538, WO 2004/101539 and WO 02/47671 (including LY-450139).

[0277] A compound of the instant invention may also be useful for treating or preventing cancer in combination with PARP inhibitors.

[0278] A compound of the instant invention may also be useful for treating cancer in combination with the following therapeutic agents: abarelix (Plenaxis depot®); aldesleukin (Prokine®); Aldesleukin (Proleukin®); Alemtuzumabb (Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole (Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine (Vidaza®); bendamustine hydrochloride (Treanda®); bevacuzimab (Avastin®); bexarotene capsules (Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); brefeldin A; busulfan intravenous (Busulfex®); busulfan oral (Myleran®); calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®); carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®); dalteparin sodium injection (Fragmin®); Darbepoetin alfa (Aranesp®); dasatinib (Sprycel®); daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); degarelix (Finnagon®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®); dexrazoxane hydrochloride (Totect®); didemnin B; 17-DMAG; docetaxel (Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal (Doxil®); dromostanolone propionate (Dromostanolone ®); dromostanolone propionate (Masterone Injection®); eculizumab injection (Soliris®); Elliott's B Solution (Elliott's B Solution®); eltrombopag (Promacta®); epirubicin (Ellence®); Epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine (Emcyt®); ethinyl estradiol; etoposide phosphate (Etopophos®); etoposide, VP- 16 (Vepesid®); everolimus tablets (Afinitor®); exemestane (Aromasin®); ferumoxytol (Feraheme Injection®); Filgrastim (Neupogen®); floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®); gefitinib (Iressa®); geldanamycin; gemcitabine (Gemzar®); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate (Zoladex Implant®); goserelin acetate (Zoladex®); histrelin acetate (Histrelin implant®); hydroxyurea (Hydrea®); Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide (IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a (Roferon A®); Interferon alfa-2b (Intron A®); iobenguane I 123 injection (AdreView®); irinotecan (Camptosar®); ixabepilone (Ixempra®); lapatinib tablets (Tykerb®); lenalidomide (Revlimid®); letrozole (Femara®); leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate (Eligard®); levamisole (Ergamisol®); lomustine, CCNU (CeeBU®); meclorethamine, nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan, L- PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnex tabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); 8-methoxypsoralen; mitomycin C (Mutamycin®); mitotane (Lysodren®); miloxantrone (Novantrone®); mitramycin; nandrolone phenpropionate (Durabolin-50®); nelarabine (Arranon®); nilotinib (Tasigna®); Nofetumomab (Verluma®); ofatumumab (Arzerra®); Oprelvekin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®); panitumumab (Vectibix®); pazopanib tablets (Votrienttm®); pegademase (Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®); pemetrexed disodium (Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plerixafor (Mozobil®); plicamycin, mithramycin (Mithracin®); porfimer sodium (Photofrin®); pralatrexate injection (Folotyn®); procarbazine (Matulane®); quinacrine (Atabrine®); rapamycin; Rasburicase (Elitek®); raloxifene hydrochloride (Evista®); Rituximab (Rituxan®); romidepsin (Istodax®); romiplostim (Nplate®); sargramostim (Leukine®); Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®); temozolomide (Temodar®); temsirolimus (Torisel®); teniposide, VM-26 (Vumon®); testolactone (Teslac®); thioguanine, 6-TG (Thiogiianine®); thiopurine; thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab (Bexxar®); Tositumomab/I-131 tositumomab (Bexxar®); trans- retinoic acid; Trastuzumab (Herceptin®); tretinoin, ATRA (Vesanoid®); triethylenemelamine; Uracil Mustard (Uracil Mustard Capsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine (Oncovin®); vinorelbine (Navelbine®); vorinostat (Zolinza®); wortmannin; and zoled onate (Zometa®).

[0279] The invention also provides a combination comprising an siNA molecule of the invention comprising at least a 15 nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 1210, SEQ ID NO: 2, SEQ ID NO: 121 1 , SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO: 1213; or formula (A) and/or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof together with another MAPK1 inhibitor.

[0280] The combinations referred to above can conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical compositions comprising a combination as defined above together with a pharmaceutically acceptable diluent or carrier represent a further aspect of the invention.

[0281] The individual compounds of such combinations can be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. In one embodiment, the individual compounds will be administered simultaneously in a combined pharmaceutical formulation.

[0282] Thus, the described molecules could be used in combination with one or more known compounds, treatments, or procedures to prevent or treat diseases, disorders, conditions, and traits described herein in a subject or organism as are known in the art, such as other MAPK1 inhibitors.

3. Therapeutic Applications

[0283] The present body of knowledge in MAPK1 research indicates the need for methods that can regulate MAPK1 expression for therapeutic use.

[0284] Thus, one aspect of the invention comprises a method of treating a subject including, but not limited to, a human suffering from a condition which is mediated by the action, or by loss of action, of MAPK1 gene expression, which method comprises administering to said subject an effective amount of a double-stranded siNA molecule of the invention. In one embodiment of this aspect, the siNA molecules comprises at least a 15 nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 1210, SEQ ID NO: 2, SEQ ID NO: 121 1 , SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO: 1213; or formula (A). In another embodiment of this aspect, the condition is or is caused by cancer. Thus, in certain embodiments the molecules and compositions of the instant invention are useful in a method for treating cancer. Cancers treatable according to this aspect of the invention include bladder cancer, bladder transitional cell carcinoma, urothelial carcinoma, brain cancer, gliomas, astrocytomas, breast cancer, breast carcinoma, cervical cancer, colorectal cancer, rectal cancer, colorectal carcinoma, colon cancer, hereditary nonpolyposis colorectal cancer, endometrial carcinoma, esophageal cancer, esophageal squamous cell carcinoma, ocular melanoma, uveal melanoma, intraocular melanoma, primary intraocular lymphoma, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, leukemia, acute lymocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid (CML), chronic myelomonocytic (CMML), liver cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, lung cancer, small cell lung cancer (SCLC), non- small cell lung cancer (NSCLC), Non-Hodgkin lymphoma, B-cell lymphomas, T-cell lymphomas, precursor T-lymphoblastic lymphoma/leukemia, multiple myeloma, ovarian carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, mucosal melanoma, small intestine carcinomas, stomach cancer, gastric carcinoma, testicular cancer, testicular seminomas, testicular non- seminomas, thyroid cancer, papillary thyroid carcinoma, and papillary adenocarcinomas.

[0285] In one embodiment, the siNA molecules of the instant invention are useful in a method for treating or preventing cancer selected from: brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, renal cell carcinoma, leukemia, hepatocellular carcinoma, lung cancer, b-cell lymphomas, ovarian carcinoma, pancreatic cancer, prostate cancer, melanoma, and stomach cancer. In certain embodiments, the compounds of the instant invention are useful for treating colorectal cancer, hepatocellular carcinoma, lung cancer, pancreatic cancer, and melanoma. In a particular embodiment, the compounds of the instant invention are useful for treating hepatocellular carcinoma.

[0286] In another embodiment, the siNA molecules of the instant invention are useful in a method for the prevention or modulation of the metastases of cancer cells and cancer. In particular, the siNA molecules of the instant invention are useful in a method to prevent or modulate the metastases of brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, renal cell carcinoma, leukemia, hepatocellular carcinoma, lung cancer, b-cell lymphomas, ovarian carcinoma, pancreatic cancer, prostate cancer, melanoma, and stomach cancer.

[0287] In certain embodiments, the administration of the siNA molecule is via local administration or systemic administration. In other embodiments, the invention features contacting the subject or organism with an siNA molecule of the invention via local administration to relevant tissues or cells, such as lung cells and tissues, such as via pulmonary delivery. In yet other embodiments, the invention features contacting the subject or organism with an siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as cancerous tissues or cells in a subject or organism.

[0288] siNA molecules of the invention are also used as reagents in ex vivo applications. For example, siNA reagents are introduced into tissue or cells that are transplanted into a subject for therapeutic effect. The cells and/or tissue can be derived from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation. The siNA molecules can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo. In one embodiment, certain MAPK1 target cells from a patient are extracted. These extracted cells are contacted with MAPK1 siNAs targeting a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells (e.g., using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells). The cells are then reintroduced back into the same patient or other patients.

[0289] For therapeutic applications, a pharmaceutically effective dose of the siNA molecules or pharmaceutical compositions of the invention is administered to the subject. A pharmaceutically effective dose is that dose required to prevent, inhibit the occun-ence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state. One skilled in the art can readily determine a therapeutically effective dose of the siNA of the invention to be administered to a given subject, by taking into account factors, such as the size and weight of the subject, the extent of the disease progression or penetration, the age, health, and sex of the subject, the route of administration, and whether the administration is regional or systemic. Generally, an amount between 0.1 μg kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The siNA molecules of the invention can be administered in a single dose or in multiple doses.

[0290] siNA molecules of the instant invention can be administered once monthly, once weekly, once daily (QD), or divided into multiple monthly, weekly, or daily doses, such as, for example, but not limitation, twice daily (BID), three times daily (TID), once every two weeks. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.

[0291] In addition, the administration can be continuous, i.e., every day, or intermittently. For example, intermittent administration of a compound of the instant invention may be administration one to six days per week or it may mean administration in cycles (e.g. daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days.

G. Administration

[0292] Compositions or formulations can be administered in a variety of ways. Non-limiting examples of administration methods of the invention include oral, buccal, sublingual, parenteral (i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly), local rectal administration or other local administration. In one embodiment, the composition of the invention can be administered by insufflation and inhalation. Administration can be accomplished via single or divided doses. In some embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection (see, e.g., U.S. Pat. No. 5,286,634). Lipid nucleic acid particles can be administered by direct injection at the site of disease or by injection at a site distal from the site of disease (see, e.g., Culver, HUMAN GENE THERAPY, Mary Ann Liebert, Inc., Publishers, New York. pp. 70-71(1994)). In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to a cell, subject, or organism as is described herein and as is generally known in the art.

1. In Vivo Administration

[0293] In any of the methods of treatment of the invention, the siNA can be administered to the subject systemically as described herein or otherwise known in the art, either alone as a monotherapy or in combination with additional therapies described herein or as are known in the art. Systemic administration can include, for example, pulmonary (inhalation, nebulization etc.) intravenous, subcutaneous, intramuscular, catheterization, nasopharangeai, transdermal, or oral/gastrointestinal administration as is generally known in the art.

[0294J In any of the methods of treatment or prevention of the invention, the siNA can be administered to the subject locally or to local tissues as described herein or otherwise known in the art, either alone as a monotherapy or in combination with additional therapies as are known in the art. Local administration can include, for example, inhalation, nebulization, catheterization, implantation, direct injection, dermal/transdermal application, patches, stenting, ear/eye drops, or portal vein administration to relevant tissues, or any other local administration technique, method or procedure, as is generally known in the art.

[0295] In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to the liver as is generally known in the art (see for example Wen et al, 2004, World J Gastroenterol., 10, 244-9; Murao et al, 2002, Pharm Res., 19, 1808-14; Liu et a!., 2003, gene Ther., 10, 180-7; Hong et al, 2003, J Pharm Pharmacol., 54, 51 -8; Herrmann et al., 2004, Arch Virol, 149, 161 1-7; and Matsuno et al, 2003, gene Ther., 10, 1559-66).

[0296] In one embodiment, the invention features the use of methods to deliver the siNA molecules of the instant invention to hematopoietic cells, including monocytes and lymphocytes. These methods are described in detail by Hartmann et al, 1998, J. Phamacol. Exp. Ther., 285(2), 920-928; Kronenwett et al, 1998, Blood, 91(3), 852-862; Filion and Phillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei, 1996, Leuk. Res., 20(1 1 /12), 925-930; and Bongartz et o/., 1994, Nucleic Acids Research, 22(22), 4681 -8.

[0297] In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered directly or topically (e.g., locally) to the dermis or follicles as is generally known in the art (see for example Brand, 2001 , Curt: Opin. Mol. Ther., 3, 244-8; Regnier el al., 1998, J. Drug Target, 5, 275-89; anikkannan, 2002, BioDrugs, 16, 339-47; Wraight et al., 2001 , Pharmacol. Ther., 90, 89-104; and Preat and Dujardin, 2001 , STP PharmaSciences, 1 1 , 57-68). In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered directly or topically using a hydroalcoholic gel formulation comprising an alcohol (e.g., ethanol or isopropanol), water, and optionally including additional agents such isopropyl myristate and carbomer 980. In other embodiments, the siNA are formulated to be administered topically to the nasal cavity. Topical preparations can be administered by one or more applications per day to the affected area; over skin areas occlusive dressings can advantageously be used. Continuous or prolonged delivery can be achieved by an adhesive reservoir system.

[0298] In one embodiment, an siNA molecule of the invention is administered iontophoretically, for example to a particular organ or compartment (e.g., the eye, back of the eye, heart, liver, kidney, bladder, prostate, tumor, CNS etc.). Non-limiting examples of iontophoretic delivery are described in, for example, WO 03/043689 and WO 03/030989, which are incorporated by reference in their entireties herein.

[0299] In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to the lung as is described herein and as is generally known in the art. In another embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to lung tissues and cells as is described in U.S. Patent Publication Nos. 2006/0062758; 2006/0014289; and 2004/0077540.

2. Aerosols and Delivery Devices a. Aerosol Formulations [0300] The compositions of the present invention, either alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation (e.g., intranasally or intratracheal ly) (see, Brigham et al., Am. J. ScL, 298:278 (1989)). Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0301] In one embodiment, the siNA molecules of the invention and formulations thereof are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues. Solid particulate compositions containing respirable dry particles of micronized nucleic acid compositions can be prepared by grinding dried or lyophilized nucleic acid compositions, and then passing the micronized composition through, for example, a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprising the siNA compositions of the invention can optionally contain a dispersant which serves to facilitate the formation of an aerosol as well as other therapeutic compounds. A suitable dispersant is lactose, which can be blended with the nucleic acid compound in any suitable ratio, such as a 1 to 1 ratio by weight.

[0302] Spray compositions comprising siNA molecules or compositions of the invention can, for example, be formulated as aqueous solutions or suspensions or as aerosols delivered from pressurized packs, such as a metered dose inhaler, with the use of a suitable liquefied propellant. In one embodiment, aerosol compositions of the invention suitable for inhalation can be either a suspension or a solution and generally contain an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 1210, SEQ ID NO: 2, SEQ ID NO: 121 1 , SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO: 1213; or formula (A), and a suitable propellant such as a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes, especially 1 ,1 , 1 ,2-tetrafluoroethane, 1 ,1 ,1 , 2,3, 3,3-heptafluoro-n-propane or a mixture thereof. The aerosol composition can optionally contain additional formulation excipients well known in the art such as surfactants. Non-limiting examples include oleic acid, lecithin or an oligolactic acid or derivative such as those described in W094/21229 and W098/34596 and co-solvents for example ethanol. In one embodiment a pharmaceutical aerosol formulation of the invention comprising a compound of the invention and a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof as propellant, optionally in combination with a surfactant and/or a co-solvent.

[0303] The aerosol formulations of the invention can be buffered by the addition of suitable buffering agents.

[0304] Aerosol formulations can include optional additives including preservatives if the formulation is not prepared sterile. Non-limiting examples include, methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants. In one embodiment, fluorocarbon or perfluorocarbon carriers are used to reduce degradation and provide safer biocompatible non-liquid particulate suspension compositions of the invention (e.g., siNA and/or LNP formulations thereof). In another embodiment, a device comprising a nebulizer delivers a composition of the invention (e.g., siNA and/or LNP formulations thereof) comprising fluorochemicals that are bacteriostatic thereby decreasing the potential for microbial growth in compatible devices.

[0305] Capsules and cartridges comprising the composition of the invention for use in an inhaler or insufflator, of for example gelatine, can be formulated containing a powder mix for inhalation of a compound of the invention and a suitable powder base such as lactose or starch. In one embodiment, each capsule or cartridge contains an siNA molecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 1210, SEQ ID NO: 2, SEQ ID NO: 121 1 , SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO:1213; or formula (A), and one or more excipients. In another embodiment, the compound of the invention can be presented without excipients such as lactose

[0306] The aerosol compositions of the present invention can be administered into the respiratory system as a formulation including particles of respirable size, e.g. particles of a size sufficiently small to pass through the nose, mouth and larynx upon inhalation and through the bronchi and alveoli of the lungs. In general, respirable particles range from about 0.5 to 10 microns in size. In one embodiment, the particulate range can be from 1 to 5 microns. In another embodiment, the particulate range can be from 2 to 3 microns. Particles of non- respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is thus minimized. For nasal administration, a particle size in the range of 10-500 um is preferred to ensure retention in the nasal cavity.

[0307} In some embodiments, an siNA composition of the invention is administered topically to the nose for example, for the treatment of rhinitis, via pressurized aerosol formulations, aqueous formulations administered to the nose by pressurized pump or by nebulization. Suitable formulations contain water as the diluent or carrier for this purpose. In certain embodiments, the aqueous formulations for administration of the composition of the invention to the lung or nose can be provided with conventional excipients such as buffering agents, tonicity modifying agents and the like. b. Devices

[0308] The siNA molecules of the invention can be formulated and delivered as particles and/or aerosols as discussed above and dispensed from various aerosolization devices known by those of skill in the art.

[0309] Aerosols of liquid or non-liquid particles comprising an siNA molecule or formulation of the invention can be produced by any suitable means, such as with a device comprising a nebulizer (see for example U.S. Pat. 4,501 ,729) such as ultrasonic or air jet nebulizers.

[0310] Solid particle aerosols comprising an siNA molecule or formulation of the invention and surfactant can be produced with any solid particulate aerosol generator. One type of solid particle aerosol generator used with the siNA molecules of the invention is an insufflator. A second type of illustrative aerosol generator comprises a metered dose inhaler ("MDF). MDIs containing siNA molecules or formulations taught herein can be prepared by methods of the art (for example, see Byron, above and WO96/32099).

[0311] The siNA molecules can also be formulated as a fluid formulation for delivery from a fluid dispenser, such as those described and illustrated in WO05/044354.

[0312] In certain embodiments of the invention, nebulizer devices are used in applications for conscious, spontaneously breathing subjects, and for controlled ventilated subjects of all ages. The nebulizer devices can be used for targeted topical and systemic drug delivery to the lung. In one embodiment, a device comprising a nebulizer is used to deliver an siNA molecule or formulation of the invention locally to lung or pulmonary tissues. In another embodiment, a device comprising a nebulizer is used to deliver a an siNA molecule or formulation of the invention systemically.

H. Other Applications/Uses of siNA Molecules of the Invention

[0313] The siNA molecules of the invention can also be used for diagnostic applications, research applications, and/or manufacture of medicants.

[0314] In one aspect, the invention features a method for diagnosing a disease, trait, or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease, trait, or condition in the subject.

[0315] In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of MAPK l proteins arising from haplotype polymorphisms that are associated with a trait, disease or condition in a subject or organism. Analysis of MAPKl genes, or MAPKl protein or RNA levels can be used to identify subjects with such polymorphisms or those subjects who are at risk of developing traits, conditions, or diseases described herein. These subjects are amenable to treatment, for example, treatment with siNA molecules of the invention and any other composition useful in treating diseases related to target gene expression. As such, analysis of MAPKl protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of MAPKl protein or RNA levels can be used to predict treatment outcome and to detennine the efficacy of compounds and compositions that modulate the level and/or activity of certain MAPKl proteins associated with a trait, disorder, condition, or disease.

[0316] In another embodiment, the invention comprises use of a double-stranded nucleic acid according to the invention for use in the manufacture of a medicament. In an embodiment, the medicament is for use in treating a condition that is mediated by the action, or by loss of action, of MAPKl . In one embodiment, the medicament is for use in the treatment of cancer. In an embodiment, the medicament is for use in the treatment of brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, renal cell carcinoma, leukemia, hepatocellular carcinoma, lung cancer, b-cell lymphomas, ovarian carcinoma, pancreatic cancer, prostate cancer, melanoma, and stomach cancer. In a particular embodiment, the compounds of the instant invention are useful for treating hepatocellular carcinoma.

[0317] In certain embodiments, siNAs wherein at least one strand comprises at least a 1 5 nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 1210, SEQ ID NO: 2, SEQ ID NO: 121 1, SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO:1213; or formula (A),are for use in a method for treating a cancer, such as, for example but not limitation,brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, renal cell carcinoma, leukemia, hepatocellular carcinoma, lung cancer, b-cell lymphomas, ovarian carcinoma, pancreatic cancer, prostate cancer, melanoma, and stomach cancer.

I. Examples

[0318] The invention will now be illustrated with the following non-limiting examples. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results.

Example 1 : Design, Synthesis, and Identification of siNAs Active Against MAPK1. MAPKl siNA Synthesis

[0319] A series of siNA molecules were designed, synthesized and evaluated for efficacy against MAPKl gene expression. Certain MAPKl sequences were designed and selected by methods set forth in U.S. Application No. 60/182,605. Other sequences were designed and selected using a proprietary algorithm. The primary criteria for design of certain of the MAPKl sequences for human siNAs were (i) homology between two species (human and rhesus monkey) and (ii) high efficacy scores as determined by a proprietary algorithm. The effects of the siNAs on MAPKl RNA levels. The target sequences of the siNAs that were selected are set forth in Table la (target sequences). The sense and antisense strands of the siNA sequences corresponding to the target sequences in Table la are set forth in Table lb. Various chemically modified siNAs that were synthesized are set forth in Table lc. Table la: MAPKl Target Sequences, noting human target sites.

UGCUAGAUUCCAGCCAGGA 1292 41

CCAAGUCCAUUGAUAUUUG 856 42

AGCAAAUGAAAGAUGUAUA 526 43

UGAAUUGUAUAAUAAAUUU 994 44

GAAUUGUAUAAUAAAUUUA 995 45

UUUGAGCACCAGACCUACU 415 46

GACAUGAGAACAUCAUUGG 476 47

CUUCAGACAUGAGAACAUC 471 48

AGACAUGAGAACAUCAUUG 475 49

GCUUCAGACAUGAGAACAU 470 50

CAGACAUGAGAACAUCAUU 474 51

AAGAUGUAUAUAUAGUACA 536 52

UUACUGCGCUUCAGACAUG 463 53

CCCAAAUGCUGACUCCAAA 1077 54

UGCGCUUCAGACAUGAGAA 467 55

GCGCUUCAGACAUGAGAAC 468 56

ACUGCGCUUCAGACAUGAG 465 57

CUUACUGCGCUUCAGACAU 462 58

UUACAAGCUCUUGAAGACA 576 59

AUCUUACUGCGCUUCAGAC 460 60

UACAAGCUCUUGAAGACAC 577 61

AUCCAUUCAGCUAACGUUC 658 62

GAAGAGACUGCUAGAUUCC 1285 63

AAAGAUGUAUAUAUAGUAC 535 64

GCCCGUGUUGCAGAUCCAG 751 65

UGAAAGAUGUAUAUAUAGU 533 66

GAAAGAUGUAUAUAUAGUA 534 67

UUCCCAAAUGCUGACUCCA 1075 68

AAUUAUGUUGAAUUCCAAG 831 69

ACCAAGUCCAUUGAUAUUU 856 70

GAACAGGCUCUGGCCCACC 1 153 71

CCAAAUGCUGACUCCAAAG 1078 72

CAAAUGCUGACUCCAAAGC 1079 73

UGAUCUCAAGAUCUGUGAC 723 74

AGACUGCUAGAUUCCAGCC 1289 75

UAACGUUCUGCACCGUGAC 669 76

CUAACGUUCUGCACCGUGA 668 77

UUUACAAGCUCUUGAAGAC 575 78

UCAGCUAACGUUCUGCACC 664 79

CAUGAGAACAUCAUUGGAA 478 80

CCCAUAUCUGGAGCAGUAU 1 170 81

ACACCAAGUCCAUUGAUAU 854 82

AUGAGAACAUCAUUGGAAU 479 83 CAUCGAGCAAAUGAAAGAU 522 84

UACAGGGCUCCAGAAAUUA 817 85

ACAGGGCUCCAGAAAUUAU 818 86

GAACAGGCUGUUCCCAAAU 1065 87

CUGGAGCAGUAUUACGACC 1177 88

CUUUGGCCUGGCCCGUGUU 741 89

UCAACAAAGUUCGAGUAGC 377 90

GGCUCUGGCCCACCCAUAU 1 158 91

AUAUUUGGUCUGUAGGCUG 869 92

AGAGGAUUGAAGUAGAACA 1 139 93

GCUCUGGCCCACCCAUAUC 1159 94

CAUGAUCACACAGGGUUCC 772 95

CUCUGGCCCACCCAUAUCU 1 160 96

CAUUGAUAUUUGGUCUGUA 864 97

AUUGAUAUUUGGUCUGUAG 865 98

AUCAUUGGAAUCAAUGACA 487 99

UGCAGAUCCAGACCAUGAU 759 100

GCAGAUCC AGACC AU GAUC 760 101

AAGAGGAUUGAAGUAGAAC 1138 102

GGCCUGGCCCGUGUUGCAG 745 103

UUGCAGAUCCAGACCAUGA 758 104

CCAUGAUCACACAGGGUUC 771 105

CACCAAGUCCAUUGAUAUU 855 106

ACCCAUAUCUGGAGCAGUA 1169 107

CUCAAGCCUUCCAACCUGC 688 108

CAAGAGGAUUGAAGUAGAA 1 137 109

AUCUUUACAAGCUCUUGAA 572 1 10

UCCAGACCAUGAUCACACA 765 1 1 1

AGGGCUCCAGAAAUUAUGU 820 1 12

CUCCAAAGCUCUGGACUUA 1089 113

UGAUCACACAGGGUUCCUG 774 1 14

GAGGAUUGAAGUAGAACAG 1140 1 15

AGACCUGAAUUGUAUAAUA 990 1 16

AACAGGCUGUUCCCAAAUG 1066 1 17

CGUGUUGCAGAUCCAGACC 754 118

UGAAGACACAACACCUCAG 587 1 19

ACCAUGAUCACACAGGGUU 770 120

UCAUUGGAAUCAAUGACAU 488 121

UGAAGU AGAAC AGGCUCUG 1 146 122

UCUUUACAAGCUCUUGAAG 573 123

AUUGAAGUAGAACAGGCUC 1 144 124

CAUGGAACAGGCUGUUCCC 1061 125

UGAGAACAUCAUUGGAAUC 480 126 UUGAAGACACAACACCUCA 586 127

ACUCCAAAGCUCUGGACUU 1088 128

GCUAGGAACUAUUUGCUUU 1018 129

UAGGAACUAUUUGCUUUCU 1020 130

ACAGGCUGUUCCCAAAUGC 1067 131

AGGCUGUUCCCAAAUGCUG 1069 132

GGCUGUUCCCAAAUGCUGA 1070 133

CCAAAGCUCUGGACUUAUU 1091 134

GCUCUGGACUUAUUGGACA 1096 135

CUCUGGACUUAUUGGACAA 1097 136

UCUGG ACU U AUUGGAC AA A 1098 137

UGUUGACAUUCAACCCACA 11 18 138

AACCCACACAAGAGGAUUG 1 129 139

CCCACACAAGAGGAUUGAA 1 131 140

CACACAAGAGGAUUGAAGU 1 133 141

ACAAGAGGAUUGAAGUAGA 1136 142

CACCCAUAUCUGGAGCAGU 1 168 143

AUCUGGAGCAGUAUUACGA 1 175 144

GAGCAGUAUUACGACCCGA 1180 145

AUUACGACCCGAGUGACGA 1 187 146

CGCCGAAGCACCAUUCAAG 1212 147

UGGAUGACUUGCCUAAGGA 1244 148

GGAUGACUUGCCUAAGGAA 1245 149

AGAGACUGCUAGAUUCCAG 1287 150

AUGGUGUGCUCUGCUUAUG 352 151

GGUGUGCUCUGCUUAUGAU 354 152

UGUGCUCUGCUUAUGAUAA 356 153

GUGCUCUGCUUAUGAUAAU 357 154

UGCUCUGCUUAUGAUAAUG 358 155

GCUCUGCUUAUGAUAAUGU 359 156

UCUGCUUAUGAUAAUGUCA 361 157

UGCUUAUGAUAAUGUCAAC 363 158

AUGAUAAUGUCAACAAAGU 368 159

CCAGAGAACCCUGAGGGAG 435 160

AGAGAACCCUGAGGGAGAU 437 161

UUCAGACAUGAGAACAUCA 472 162

AUUGGAAUCAAUGACAUUA 490 163

UUGGAAUCAAUGACAUUAU 491 164

UGGAAUCAAUGACAUUAUU 492 165

AAUCAAUGACAUUAUUCGA 495 166

CAAUGACAUUAUUCGAGCA 498 167

UGACAUUAUUCGAGCACCA 501 168

GCACCAACCAUCGAGCAAA 514 169 CACCAACCAUCGAGCAAAU 515 170

CAACCAUCGAGCAAAUGAA 518 171

AUCGAGCAAAUGAAAGAUG 523 172

UCGAGCAAAUGAAAGAUGU 524 173

CGAGCAAAUGAAAGAUGUA 525 174

GAUGUAUAUAUAGUACAGG 538 175

UGUAU AUAUAGUACA GGAC 540 176

UAUAUAUAGUACAGGACCU 542 177

UACAGGACCUCAUGGAAAC 551 178

ACAGGACCUCAUGGAAACA 552 179

AGGACCUCAUGGAAACAGA 554 180

AAACAGAUCUUUACAAGCU 566 181

AACAGAUCUUUACAAGCUC 567 182

ACAGAUCUUUACAAGCUCU 568 183

ACAAGCUCUUGAAGACACA 578 184

ACAACACCUCAGCAAUGAC 594 185

ACCAGAUCCUCAGAGGGUU 632 186

CCAGAUCCUCAGAGGGUUA 633 187

UCAACACCACCUGUGAUCU 710 188

CCUGUGAUCUCAAGAUCUG 719 189

CUGUGAUCUCAAGAUCUGU 720 190

AUCUCAAGAUCUGUGACUU 725 191

GGCCCGUGUUGCAGAUCCA 750 192

ACAGGGUUCCUGACAGAAU 781 193

CAGGGUUCCUGACAGAAUA 782 194

AGGGUUCCUGACAGAAUAU 783 195

CUGACAGAAUAUGUGGCCA 790 196

GUACAGGGCUCCAGAAAUU 816 197

GAAAUUAUGUUGAAUUCCA 829 198

AUGUUGAAUUCCAAGGGCU 835 199

GUUGAAUUCCAAGGGCUAC 837 200

CAAGUCCAUUGAUAUUUGG 858 201

GCUGCAUUCUGGCAGAAAU 884 202

CAGAAAUGCUUUCUAACAG 896 203

GAAAUGCUUUCUAACAGGC 898 204

AAUGCUUUCUAACAGGCCC 900 205

GAAGCAUUAUCUUGACCAG 930 206

CAAGAAGACCUGAAUUGUA 985 207

AAGAAGACCUGAAUUGUAU 986 208

AAGACCUGAAUUGUAUAAU 989 209

GACCUGAAUUGUAUAAUAA 991 210

ACCUGAAUUGUAUAAUAAA 992 21 1

ACAACAGACUUUAAUGUAA 291 212 AGACCAUGAUCACACAGGG 768 213

CCAUUCAGCUAACGUUCUG 660 214

GCUACACCAACCUCUCGUA 31 1 215

ACACCAACCUCUCGUACAU 314 216

CCAUCGAGCAAAUGAAAGA 521 217

UGCUUUCUCUUCCACACAA 1031 218

UUGACAUUCAACCCACACA 1120 219

CAUUCAACCCACACAAGAG 1124 220

GAUUGAAGUAGAACAGGCU 1143 221

GGCCCACCCAUAUCUGGAG 1 164 222

UGACGAGCCCAUCGCCGAA 1200 223

UGGUGUGCUCUGCUUAUGA 353 224

GUGUGCUCUGCUUAUGAUA 355 225

CUGCUUAUGAUAAUGUCAA 362 226

ACAUGAGAACAUCAUUGGA 477 227

CAUUGGAAUCAAUGACAUU 489 228

GAAUCAAUGACAUUAUUCG 494 229

UCAAUGACAUUAUUCGAGC 497 230

ACAUUAUUCGAGCACCAAC 503 231

GCAAAUGAAAGAUGUAUAU 528 232

AGUACAGGACCUCAUGGAA 549 233

ACCUCAUGGAAACAGAUCU 557 234

UGGAAACAGAUCUUUACAA 563 235

GGAAACAGAUCUUUACAAG 564 236

GAAACAGAUCUUUACAAGC 565 237

UCUUGAAGACACAACACCU 584 238

ACCUCAGCAAUGACCAUAU 599 239

CCUCAAGCCUUCCAACCUG 687 240

CUUCCAACCUGCUGCUCAA 695 241

UGCUCAACACCACCUGUGA 707 242

ACACCACCUGUGAUCUCAA 713 243

CCGUGUUGCAGAUCCAGAC 753 244

CACACAGGGUUCCUGACAG 778 245

GGGCUACACCAAGUCCAUU 849 246

CAUUCUGGCAGAAAUGCUU 888 247

CUGGCAGAAAUGCUUUCUA 892 248

UGGC AGAAAUGCU UUCUAA 893 249

GGCAGAAAUGCUUUCUAAC 894 250

GCAGAAAUGCUUUCUAACA 895 251

CUAACAGGCCCAUCUUUCC 908 252

AUCACAAGAAGACCUGAAU 981 253 Table lb. Various c-MAPKl siNA sense and antisense sequences corresponding to the identified target sequences in Table la.

833 39 UUAUGUUGAAUUCCAAGGG CCCUUGGAAUUCAACAUAA 1248

832 40 AUUAUGUUGAAUUCCAAGG CCUUGGAAUUCAACAUAAU 1249

1292 41 UGCUAGAUUCCAGCCAGGA UCCUGGCUGGAAUCUAGCA 1250

856 42 CCAAGUCCAUUGAUAUUUG CAAAUAUCAAUGGACUUGG 1251

526 43 AGCAAAUGAAAGAUGUAUA UAUACAUCUUUCAUUUGCU 1252

994 44 UGAAUUGUAUAAUAAAUUU AAAUUUAUUAUACAAUUCA 1253

995 45 GAAUUGUAUAAUAAAUUUA UAAAUUUAUUAUACAAUUC 1254

415 46 UUUGAGCACCAGACCUACU AGUAGGUCUGGUGCUCAAA 1255

476 47 GACAUGAGAACAUCAUUGG CCAAUGAUGUUCUCAUGUC 1256

471 48 CUUCAGACAUGAGAACAUC GAUGUUCUCAUGUCUGAAG 1257

475 49 AGACAUGAGAACAUCAUUG CAAUGAUGUUCUCAUGUCU 1258

470 50 GCUUCAGACAUGAGAACAU AUGUUCUCAUGUCUGAAGC 1259

474 51 CAGACAUGAGAACAUCAUU AAUGAUGUUCUCAUGUCUG 1260

536 52 AAGAUGUAUAUAUAGUACA UGUACUAUAUAUACAUCUU 1261

463 53 UUACUGCGCUUCAGACAUG C AUGU CUGAAGCGCAGU AA 1262

1077 54 CCCAAAUGCUGACUCCAAA UUUGGAGUCAGCAUUUGGG 1263

467 55 UGCGCUUCAGACAUGAGAA UUCUCAUGUCUGAAGCGCA 1264

468 56 GCGCUUCAGACAUGAGAAC GUUCUCAUGUCUGAAGCGC 1265

465 57 ACUGCGCUUCAGACAUGAG CUCAUGUCUGAAGCGCAGU 1266

462 58 CUUACUGCGCUUCAGACAU AUGUCUGAAGCGCAGUAAG 1267

576 59 UUACAAGCUCUUGAAGACA UGUCUUCAAGAGCUUGUAA 1268

460 60 AUCUUACUGCGCUUCAGAC GUCUGAAGCGCAGUAAGAU 1269

577 61 UACAAGCUCUUGAAGACAC GUGUCUUCAAGAGCUUGUA 1270

658 62 AUCCAUUCAGCUAACGUUC GAACGUUAGCUGAAUGGAU 1271

1285 63 GAAGAGACUGCUAGAUUCC GGAAUCUAGCAGUCUCUUC 1272

535 64 AAAGAUGUAUAUAUAGUAC GUACUAUAUAUACAUCUUU 1273

751 65 GCCCGUGUUGCAGAUCCAG CUGGAUCUGCAACACGGGC 1274

533 66 UGAAAGAUGUAUAUAUAGU ACUAUAUAUACAUCUUUCA 1275

534 67 GAAAGAUGUAUAUAUAGUA UACUAUAUAUACAUCUUUC 1276

1075 68 UUCCCAAAUGCUGACUCCA UGGAGUCAGCAUUUGGGAA 1277

831 69 AAUUAUGUUGAAUUCCAAG CUUGGAAUUCAACAUAAUU 1278

856 70 ACCAAGUCCAUUGAUAUUU AAAUAUCAAUGGACUUGGU 1279

1 153 71 GAACAGGCUCUGGCCCACC GGUGGGCCAGAGCCUGUUC 1280

1078 72 CCAAAUGCUGACUCCAAAG CUUUGGAGUCAGCAUUUGG 1281

1079 73 CAAAUGCUGACUCCAAAGC GCUUUGGAGUCAGCAUUUG 1282

723 74 UGAUCUCAAGAUCUGUGAC GUCACAGAUCUUGAGAUCA 1283

1289 75 AGACUGCUAGAUUCCAGCC GGCUGGAAUCUAGCAGUCU 1284

669 76 UAACGUUCUGCACCGUGAC GUCACGGUGCAGAACGUUA 1285

668 77 CUAACGUUCUGCACCGUGA UCACGGUGCAGAACGUUAG 1286

575 78 UUUACAAGCUCUUGAAGAC GUCUUCAAGAGCUUGUAAA 1287

664 79 UCAGCUAACGUUCUGCACC GGUGCAGAACGUUAGCUGA 1288

478 80 CAUGAGAACAUCAUUGGAA UUCCAAUGAUGUUCUCAUG 1289

1 170 81 CCCAUAUCUGGAGCAGUAU AUACUGCUCCAGAUAUGGG 1290 854 82 ACACCAAGUCCAUUGAUAU AUAUCAAUGGACUUGGUGU 1291

479 83 AUGAGAACAUCAUUGGAAU AUUCCAAUGAUGUUCUCAU 1292

522 84 CAUCGAGCAAAUGAAAGAU AUCUUUCAUUUGCUCGAUG 1293

817 85 UACAGGGCUCCAGAAAUUA UAAUUUCUGGAGCCCUGUA 1294

818 86 ACAGGGCUCCAGAAAUUAU AUAAUUUCUGGAGCCCUGU 1295

1065 87 GAACAGGCUGUUCCCAAAU AUUUGGGAACAGCCUGUUC 1296

1 177 88 CUGGAGCAGUAUUACGACC GGUCGUAAUACUGCUCCAG 1297

741 89 CUUUGGCCUGGCCCGUGUU AACACGGGCCAGGCCAAAG 1298

377 90 UCAACAAAGUUCGAGUAGC GCUACUCGAACUUUGUUGA 1299

1158 91 GGCUCUGGCCCACCCAUAU AUAUGGGUGGGCCAGAGCC 1300

869 92 AUAUUUGGUCUGUAGGCUG CAGCCUACAGACCAAAUAU 1301

1 139 93 AGAGGAUUGAAGUAGAACA UGUUCUACUUCAAUCCUCU 1302

1159 94 GCUCUGGCCCACCCAUAUC GAUAUGGGUGGGCCAGAGC 1303

772 95 CAUGAUCACACAGGGUUCC GGAACCCUGUGUGAUCAUG 1304

1 160 96 CUCUGGCCCACCCAUAUCU AGAUAUGGGUGGGCCAGAG 1305

864 97 CAUUGAUAUUUGGUCUGUA UACAGACCAAAUAUCAAUG 1306

865 98 AUU GAUAUUUGGUCU GUAG CUACAGACCAAAUAUCAAU 1307

487 99 AUCAUUGGAAUCAAUGACA UGUCAUUGAUUCCAAUGAU 1308

759 100 UGCAGAUCCAGACCAUGAU AUCAUGGUCUGGAUCUGCA 1309

760 101 GCAGAUCCAGACCAUGAUC GAUCAUGGUCUGGAUCUGC 1310

1 138 102 AAGAGGAUUGAAGUAGAAC GUUCUACUUCAAUCCUCUU 131 1

745 103 GGCCUGGCCCGUGUUGCAG CUGCAACACGGGCCAGGCC 1312

758 104 UUGCAGAUCCAGACCAUGA UCAUGGUCUGGAUCUGCAA 1313

771 105 CCAUGAUCACACAGGGUU C GAACCCUGUGUGAUCAUGG 1314

855 106 CACCAAGUCCAUUGAUAUU AAUAUCAAUGGACUUGGUG 1315

1169 107 ACCCAUAUCUGGAGCAGUA UACUGCUCCAGAUAUGGGU 1316

688 108 CUCAAGCCUUCCAACCUGC GCAGGUUGGAAGGCUUGAG 1317

1137 109 C A AG AGGAUUGAAGU A GA A UUCUACUUCAAUCCUCUUG 1318

572 110 AUCUUUACAAGCUCUUGAA UUCAAGAGCUUGUAAAGAU 1319

765 1 1 1 UCCAGACCAUGAUCACACA UGUGUGAUCAUGGUCUGGA 1320

820 112 AGGGCUCCAGAAAUUAUGU ACAUAAUUUCUGGAGCCCU 1321

1089 113 CUCCAAAGCUCUGGACUUA UAAGUCCAGAGCUUUGGAG 1322

774 114 UGAUCACACAGGGUUCCUG CAGGAACCCUGUGUGAUCA 1323

1140 115 GAGGAUUGAAGUAGAACAG CUGUUCUACUUCAAUCCUC 1324

990 116 AGACCUGAAUUGUAUAAUA UAUUAUACAAUUCAGGUCU 1325

1066 1 17 AACAGGCUGUUCCCAAAUG CAUUUGGGAACAGCCUGUU 1326

754 1 18 CGUGUUGCAGAUCCAGACC GGUCUGGAUCUGCAACACG 1327

587 1 19 UGAAGACACAACACCUCAG CUGAGGUGUUGUGUCUUCA 1328

770 120 ACCAUGAUCACACAGGGUU AACCCUGUGUGAUCAUGGU 1329

488 121 UCAUUGGAAUCAAUGACAU AUGUCAUUGAUUCCAAUGA 1330

1146 122 UGAAGUAGAACAGGCUCUG CAGAGCCUGUUCUACUUCA 1331

573 123 UCUUUACAAGCUCUUGAAG CUUCAAGAGCUUGUAAAGA 1332

1 144 124 AUUGAAGUAGAACAGGCUC GAGCCUGUUCUACUUCAAU 1333 1061 125 CAUGGAACAGGCUGUUCCC GGGAACAGCCUGUUCCAUG 1334

480 126 UGAGAACAUCAUUGGAAUC GAUUCCAAUGAUGUUCUCA 1335

586 127 UUGAAGACACAACACCUCA UGAGGUGUUGUGUCUUCAA 1336

1088 128 ACUCCAAAGCUCUGGACUU AAGUCCAGAGCUUUGGAGU 1337

1018 129 GCUAGGAACUAUUUGCUUU AAAGCAAAUAGUUCCUAGC 1338

1020 130 UAGGAACUAUUUGCUUUCU AGAAAGCAAAUAGUUCCUA 1339

1067 131 ACAGGCUGUUCCCAAAUGC GCAUUUGGGAACAGCCUGU 1340

1069 132 AGGCUGUUCCCAAAUGCUG CAGCAUUUGGGAACAGCCU 1341

1070 133 GGCUGUUCCCAAAUGCUGA UCAGCAUUUGGGAACAGCC 1342

1091 134 CCAAAGCUCUGGACUUAUU AAUAAGUCCAGAGCUUUGG 1343

1096 135 GCUCUGGACUUAUUGGACA UGUCCAAUAAGUCCAGAGC 1344

1097 136 CUCUGGACUUAUUGGACAA UUGUCCAAUAAGUCCAGAG 1345

1098 137 UCUGGACUUAUUGGACAAA UUUGUCCAAUAAGUCCAGA 1346

1 1 18 138 UGUUGACAUUCAACCCACA UGUGGGUUGAAUGUCAACA 1347

1 129 139 AACCCACACAAGAGGAUUG CAAUCCUCUUGUGUGGGUU 1348

1 131 140 CCCACACAAGAGGAUUGAA UUCAAUCCUCUUGUGUGGG 1349

1133 141 CACACAAGAGGAUUGAAGU ACUUCAAUCCUCUUGUGUG 1350

1 136 142 ACAAGAGGAUUGAAGUAGA UCUACUUCAAUCCUCUUGU 1351

1168 143 CACCCAUAUCUGGAGCAGU ACUGCUCCAGAUAUGGGUG 1352

1 175 144 AUCUGGAGCAGUAUUACGA UCGUAAUACUGCUCCAGAU 1353

1180 145 GAGCAGUAUUACGACCCGA UCGGGUCGUAAUACUGCUC 1354

1187 146 AUUACGACCCGAGUGACGA UCGUCACUCGGGUCGUAAU 1355

1212 147 CGCCGAAGCACCAUUCAAG CUUGAAUGGUGCUUCGGCG 1356

1244 148 UGGAUGACUUGCCUAAGGA UCCUUAGGCAAGUCAUCCA 1357

1245 149 GGAUGACUUGCCUAAGGAA UUCCUUAGGCAAGUCAUCC 1358

1287 150 AGAGACUGCUAGAUUCCAG CUGGAAUCUAGCAGUCUCU 1359

352 151 AUGGUGUGCUCUGCUUAUG CAUAAGCAGAGCACACCAU 1360

354 152 GGUGUGCUCUGCIJUAUGAU AUCAUAAGCAGAGCACACC 1361

356 153 UGUGCUCUGCUUAUGAUAA UUAUCAUAAGCAGAGCACA 1362

357 154 GUGCUCUGCUUAUGAUAAU AUUAUCAUAAGCAGAGCAC 1363

358 155 UGCUCUGCUUAUGAUAAUG CAUUAUCAUAAGCAGAGCA 1364

359 156 GCUCUGCUUAUGAUAAUGU ACAUUAUCAUAAGCAGAGC 1365

361 157 UCUGCUUAUGAUAAUGUCA UGACAUUAUCAUAAGCAGA 1366

363 158 UGCUUAUGAUAAUGUCAAC GUUGACAUUAUCAUAAGCA 1367

368 159 AUGAUAAUGUCAACAAAGU ACUUUGUUGACAUUAUCAU 1368

435 160 CCAGAGAACCCUGAGGGAG CUCCCUCAGGGUUCUCUGG 1369

437 161 AGAGAACCCUGAGGGAGAU AUCUCCCUCAGGGUUCUCU 1370

472 162 UUCAGACAUGAGAACAUCA UGAUGUUCUCAUGUCUGAA 1371

490 163 AUUGGAAUCAAUGACAUUA UAAUGUCAUUGAUUCCAAU 1372

491 164 UIJGGAAUCAAUGACAUUAU AUAAUGUCAUUGAUUCCAA 1373

492 165 UGGAAUCAAUGACAUUAUU AAUAAUGUCAUUGAUUCCA 1374

495 166 AAUCAAUGACAUUAUUCGA UCGAAUAAUGUCAUUGAUU 1375

498 167 CAAUGACAUUAUUCGAGCA UGCUCGAAUAAUGUCAUUG 1376 501 168 UGACAUUAUUCGAGCACCA UGGUGCUCGAAUAAUGUCA 1377

514 169 GCACCAACCAUCGAGCAAA UUUGCUCGAUGGUUGGUGC 1378

515 170 C ACC AACCAU CGAGC AA AU AUUUGCUCGAUGGUUGGUG 1379

518 171 CAACCAUCGAGCAAAUGAA UUCAUUUGCUCGAUGGUUG 1380

523 172 AUCGAGCAAAUGAAAGAUG CAUCUUUCAUUUGCUCGAU 1381

524 173 UCGAGCAAAUGAAAGAUGU AC AUCUUUCAUUUGCUCGA 1382

525 174 CGAGCAAAUGAAAGAUGUA UACAUCUUUCAUUUGCUCG 1383

538 175 GAUGUAUAUAUAGUACAGG CCUGUACUAUAUAUACAUC 1384

540 176 UGUAUAUAUAGUACAGGAC GUCCUGUACUAUAUAUACA 1385

542 177 UAUAUAUAGUACAGGACCU AGGUCCUGUACUAUAUAUA 1386

551 178 UACAGGACCUCAUGGAAAC GUUUCCAUGAGGUCCUGUA 1387

552 179 ACAGGACCUCAUGGAAACA UGUUUCCAUGAGGUCCUGU 1388

554 180 AGGACCUCAUGGAAACAGA UCUGUUUCCAUGAGGUCCU 1389

566 181 AAACAGAUCUUUACAAGCU AGCUUGUAAAGAUCUGUUU 1390

567 182 AACAGAUCUUUACAAGCUC GAGCUUGUAAAGAUCUGUU 1391

568 183 ACAGAUCUUUACAAGCUCU AGAGCUUGUAAAGAUCUGU 1392

578 184 ACAAGCUCUUGAAGACACA UGUGUCUUCAAGAGCUUGU 1393

594 185 ACAACACCUCAGCAAUGAC GUCAUUGCUGAGGUGUUGU 1394

632 186 ACCAGAUCCUCAGAGGGUU AACCCUCUGAGGAUCUGGU 1395

633 187 CCAGAUCCUCAGAGGGUUA UAACCCUCUGAGGAUCUGG 1396

710 188 UCAACACCACCUGUGAUCU AGAUCACAGGUGGUGUUGA 1397

719 189 CCUGUGAUCUCAAGAUCUG CAGAUCUUGAGAUCACAGG 1398

720 190 CUGUGAUCUCAAGAUCUGU ACAGAUCUUGAGAUCACAG 1399

725 191 AUCUCAAGAUCUGUGACUU AA GUCAC AG AUCUUGAG AU 1400

750 192 GGCCCGUGUUGCAGAUCCA UGGAUCUGCAACACGGGCC 1401

781 193 ACAGGGUUCCUGACAGAAU AUUCUGUCAGGAACCCUGU 1402

782 194 CAGGGUUCCUGACAGAAUA UAUUCUGUCAGGAACCCUG 1403

783 195 AGGGUUCCUGACAGAAUAU AUAUUCUGUCAGGAACCCU 1404

790 196 CUGACAGAAUAUGUGGCCA UGGCCACAUAUUCUGUCAG 1405

816 197 GUACAGGGCUCCAGAAAUU AAUUUCUGGAGCCCUGUAC 1406

829 198 GAAAUUAUGUUGAAUUCCA UGGAAUUCAACAUAAUUUC 1407

835 199 AUGUUGAAUUCCAAGGGCU AGCCCUUGGAAUUCAACAU 1408

837 200 GUUGAAUUCCAAGGGCUAC GUAGCCCUUGGAAUUCAAC 1409

858 201 CAAGUCCAUUGAUAUUUGG CCAAAUAUCAAUGGACUUG 1410

884 202 GCUGCAUUCUGGCAGAAAU AUUUCUGCCAGAAUGCAGC 141 1

896 203 CAGAAAUGCUUUCUAACAG CUGUUAGAAAGCAUUUCUG 1412

898 204 GAAAUGCUUUCUAACAGGC GCCUGUUAGAAAGCAUUUC 1413

900 205 AAUGCUUUCUAACAGGCCC GGGCCUGUUAGAAAGCAUU 1414

930 206 GAAGCAUUAUCUUGACCAG CUGGUCAAGAUAAUGCUUC 1415

985 207 CAAGAAGACCUGAAUUGUA UACAAUUCAGGUCUUCUUG 1416

986 208 AAGAAGACCUGAAUUGUAU AUACAAUUCAGGUCUUCUU 1417

989 209 AAGACCUGAAUUGUAUAAU AUUAUACAAUUCAGGUCUU 1418

991 210 GACCUGAAUUGUAUAAUAA UUAUUAUACAAUUCAGGUC 1419 992 211 ACCUGAAUUGUAUAAUAAA UUUAUUAUACAAUUCAGGU 1420

291 212 ACAACAGACUUUAAUGUAA UUACAUUAAAGUCUGUUGU 1421

768 213 AGACCAUGAUCACACAGGG CCCUGUGUGAUCAUGGUCU 1422

660 214 CCAUUCAGCUAACGUUCUG CAGAACGUUAGCUGAAUGG 1423

31 1 215 GCUACACCAACCUCUCGUA UACGAGAGGUUGGUGUAGC 1424

314 216 ACACCAACCUCUCGUACAU AUGUACGAGAGGUUGGUGU 1425

521 217 CCAUCGAGCAAAUGAAAGA UCUUUCAUUUGCUCGAUGG 1426

1031 218 UGCUUUCUCUUCCACACAA UUGUGUGGAAGAGAAAGCA 1427

1 120 219 UUGACAUUCAACCCACACA UGUGUGGGUUGAAUGUCAA 1428

1124 220 CAUUGAACCCACACAAGAG CUCUUGUGUGGGUUGAAUG 1429

1 143 221 GAUUGAAGUAGAACAGGCU AGCCUGUUCUACUUCAAUC 1430

1164 222 GGCCCACCCAUAUCUGGAG CUCCAGAUAUGGGUGGGCC 1431

1200 223 UGACGAGCCCAUCGCCGAA UUCGGCGAUGGGCUCGUCA 1432

353 224 UGGUGUGCUCUGCUUAUGA UCAUAAGCAGAGCACACCA 1433

355 225 GUGUGCUCUGCUUAUGAUA UAUCAUAAGCAGAGCACAC 1434

362 226 CUGCUUAUGAUAAUGUCAA UUGACAUUAUCAUAAGCAG 1435

477 227 ACAUGAGAACAUCAUUGGA UCCAAUGAUGUUCUCAUGU 1436

489 228 CAUUGGAAUCAAUGACAUU AAUGUCAUUGAUUCCAAUG 1437

494 229 GAA UC AAUGAC AUUAUUCG CGAAUAAUGUCAUUGAUUC 1438

497 230 UCAAUGACAUUAUUCGAGC GCUCGAAUAAUGUCAUUGA 1439

503 231 ACAUUAUUCGAGCACCAAC GUUGGUGCUCGAAUAAUGU 1440

528 232 GCAAAUGAAAGAUGUAUAU AUAUACAUCUUUCAUUUGC 1441

549 233 AGUACAGGACCUCAUGGAA UUCCAUGAGGUCCUGUACU 1442

557 234 ACCUCAUGGAAACAGAUCU AGAUCUGUUUCCAUGAGGU 1443

563 235 UGGAAACAGAUCUUUACAA UUGUAAAGAUCUGUUUCCA 1444

564 236 GGAAACAGAUCUUUACAAG CUUGUAAAGAUCUGUUUCC 1445

565 237 GAAACAGAUCUUUACAAGC GCUUGUAAAGAUCUGUUUC 1446

584 238 UCUUGAAGACACAACACCU AGGUGUUGUGUCUUCAAGA 1447

599 239 ACCUCAGCAAUGACCAUAU AUAUGGUCAUUGCUGAGGU 1448

687 240 CCUCAAGCCUUCCAACCUG CAGGUUGGAAGGCUUGAGG 1449

695 241 CUUCCAACCUGCUGCUCAA UUGAGCAGCAGGUUGGAAG 1450

707 242 UGCUCAACACCACCUGUGA UCACAGGUGGUGUUGAGCA 1451

713 243 ACACCACCUGUGAUCUCAA UUGAGAUCACAGGUGGUGU 1452

753 244 CCGUGUUGCAGAUCCAGAC GUCUGGAUCUGCAACACGG 1453

778 245 CACACAGGGUUCCUGACAG CUGUCAGGAACCCUGUGUG 1454

849 246 GGGCUACACCAAGUCCAUU AAUGGACUUGGUGUAGCCC 1455

888 247 CAU UCUGGCAGAAAUGCUU AAGCAUUUCUGCCAGAAUG 1456

892 248 CUGGCAGAAAUGCUUUCUA UAGAAAGCAUUUCUGCCAG 1457

893 249 UGGCAGAAAUGCUUUCUAA UUAGAAAGCAUUUCUGCCA 1458

894 250 GGCAGAAAUGCUUUCUAAC GUUAGAAAGCAUUUCUGCC 1459

895 251 GCAGAAAUGCUUUCU AA C A UGUUAGAAAGCAUUUCUGC 1460

908 252 CUAACAGGCCCAUCUUUCC GGAAAGAUGGGCCUGUUAG 1461

981 253 AUCACAAGAAGACCUGAAU AUUCAGGUCUUCUUGUGAU 1462 [0320] For each oligonucleotide of a target sequence, the two individual, complementary strands of the siNA were synthesized separately using solid phase synthesis, then purified separately by reversed phase solid phase extraction (SPE). The complementary strands were annealed to form the double strand (duplex) and delivered in the desired concentration and buffer of choice.

[0321] Briefly, the single strand oligonucleotides were synthesized using phosphoramidite chemistry on an automated solid-phase synthesizer, using procedures as are generally known in the art (see for example U.S. Application No. 12/064,014). A synthesis column was packed with solid support derivatized with the first nucleoside residue (natural or chemically modified). Synthesis was initiated by detritylation of the acid labile 5'-0-dimethoxytrityl group to release the 5'-hydroxyl. A suitably protected phosphoramidite and a suitable activator in acetonitrile were delivered simultaneously to the synthesis column resulting in coupling of the amidite to the 5'-hydroxyl. The column was then washed with a solvent, such as acetonitrile. An oxidizing solution, such as an iodine solution was pumped through the column to oxidize the phosphite triester linkage P(III) to its phosphotriester P(V) analog. Unreacted 5'-hydroxyl groups were capped using reagents such as acetic anhydride in the presence of 2,6-lutidine and N- methylimidazole. The elongation cycle was resumed with the detritylation step for the next phosphoramidite incorporation. This process was repeated until the desired sequence was synthesized. The synthesis concluded with the final 5'-terminus protecting group (trityl or 5'-0- dimethoxytrityl).

[0322] Upon completion of the synthesis, the solid-support and associated oligonucleotide were dried under argon pressure or vacuum. Aqueous base was added and the mixture was heated to effect cleavage of the succinyl linkage, removal of the cyanoethyl phosphate protecting group, and deprotection of the exocyclic amine protection.

[0323] The following process was performed on single strands that do not contain ribonucleotides. After treating the solid support with the aqueous base, the mixture was filtered to separate the solid support from the deprotected crude synthesis material. The solid support was then rinsed with water, which is combined with the filtrate. The resultant basic solution allows for retention of the 5'-0-dimethoxytrityl group to remain on the 5' terminal position (trityl-on).

[0324] For single strands that contain ribonucleotides, the following process was performed. After treating the solid support with the aqueous base, the mixture was filtered to separate the solid support from the deprotected crude synthesis material. The solid support was then rinsed with dimethylsulfoxide (DMSO), which was combined with the filtrate. Fluoride reagent, such as triethylamine trihydrofluoride, was added to the mixture, and the solution was heated. The reaction was quenched with suitable buffer to provide a solution of crude single strand with the 5'-0-dimethoxytrityl group on the final 5' terminal position.

[0325] The trityl-on solution of each crude single strand was purified using chromatographic purification, such as SPE RPC purification. The hydrophobic nature of the trityl group permits stronger retention of the desired full-length oligo than the non-tritylated truncated failure sequences. The failure sequences were selectively washed from the resin with a suitable solvent, such as low percent acetonitrile. Retained oligonucleotides were then detritylated on-column with trifluoroacetic acid to remove the acid-labile trityl group. Residual acid was washed from the column, a salt exchange was performed, and a final desalting of the material commenced . The full-length oligo was recovered in a purified form with an aqueous-organic solvent. The final product was then analyzed for purity (HPLC), identity (Maldi-TOF MS), and yield (UV A₂6o)- The oligos were dried via lyophilization or vacuum condensation.

[0326] Annealing: Based on the analysis of the product, the dried oligos were dissolved in appropriate buffers followed by mixing equal molar amounts (calculated using the theoretical extinction coefficient) of the sense and antisense oligonucleotide strands. The solution was then analyzed for purity of duplex by chromatographic methods and desired final concentration. If the analysis indicated an excess of either strand, then the additional non-excess strand was titrated until duplexing was complete. When analysis indicated that the target product purity has been achieved the material was delivered and ready for use.

[0327] Below is a table showing various modified siNAs synthesized using this protocol or that can be synthesized using this protocol or using methods known in the art. Table lc: MAPKl siNA Strands Synthesized (Antisense sequences are readily identified as being complementary to the target sequence shown).

R-008352012-OOON 861 8 GUCCAUUGAUAUUUGGUCU B GUCCAUUGAUAUUUGGUCUrrB 684

R-008352012-OOON 861 8 GUCCAUUGAUAUUUGGUCU AGACCAAAUAUCAAUGGACUU 685

R-008352099-OOON 1077 54 CCCAAAUGCUGACUCCAAA B CCCAAAUGCUGACUCCAAA TT B 686

R-008352099-000N 1077 54 CCCAAAUGCUGACUCCAAA UUUGGAGUCAGCAUUUGGGUU 687

R-008352015-000P 1285 63 GAAGAGACUGCUAGAUUCC B GAAGAGACUGCUAGAUUCC7TB 688

R-008352015-OOOP 1285 63 GAAGAGACUGCUAGAUUCC GGAAUCUAGCAGUCUCUUCUU 689

R-008352018-OOOR 774 1 14 UGAUCACACAGGGUUCCUG B UGAUCACACAGGGUUCCUGJTB 690

R-008352018-OOOR 774 1 14 UGAUCACACAGGGUUCCUG CAGGAACCCUGUGUGAUCAUU 691

R-008352021-OOOX 1089 1 13 CUCCAAAGCUCUGGACUUA B CUCCAAAGCUCUGGACUUA7TB 692

R-008352021-OOOX 1089 1 13 CUCCAAAGCUCUGGACUUA UAAGUCCAGAGCUUUGGAGUU 693

R-008351985-OOOE 488 121 UCAUUGGAAUCAAUGACAU B UCAUUGGAAUCAAUGACAU7TB 694

R-008351985-OOOE 488 121 UCAUUGGAAUCAAUGACAU AUGUCAUUGAUUCCAAUGAUU 695

R-O08352102-000F 479 83 AUGAGAACAUCAUUGGAAU B AUGAGAACAUCAUUGGAAUJTB 696

R-O083521O2-0O0F 479 83 AUGAGAACAUCAUUGGAAU AUUCCAAUGAUGUUCUCAUUU 697

R-O08351958-OO0D 752 11 CCCGUGUUGCAGAUCCAGA B CCCGUGUUGCAGAUCCAGA TT B 698

R-O08351958-0O0D 752 11 CCCGUGUUGCAGAUCCAGA UCUGGAUCUGCAACACGGGUU 699

R-O08352IO5-0O0G 462 58 CUUACUGCGCUUCAGACAU B CUUACUGCGCUUCAGACAU7TB 700

R-O083521O5-000G 462 58 CUUACUGCGCUUCAGACAU AUGUCUGAAGCGCAGUAAGUU 701

R-0083521O8-000H 768 213 AGACCAUGAUCACACAGGG B AGACCAUGAUCACACAGGG7TB 702

R-008352108-OOOH 768 213 AGACCAUGAUCACACAGGG CCCUGUGUGAUCAUGGUCUUU 703

R-008351 61-OOOK 476 47 GACAUGAGAACAUCAUUGG B GACAUGAGAACAUCAUUGG TTB 704

R-008351961-OOO 476 47 GACAUGAGAACAUCAUUGG CCAAUGAUGUUCUCAUGUCUU 705

R-OO8352135-O00J 660 214 CCAUUCAGCUAACGUUCUG B CCAUUCAGCUAACGUUCUG 7TB 706

R-008352135-O OJ 660 214 CCAUUCAGCUAACGUUCUG CAGAACGUUAGCUGAAUGGUU 707

R-008351919-000T 990 1 16 AGACCUGAAUUGUAUAAUA B AGACCUGAAUUGUAUAAUA7TB 708

R-008351919-000T 990 1 16 AGACCUGAAUUGUAUAAUA UAUUAUACAAUUCAGGUCUUU 709

R-008351922-000Z 991 210 GACCUGAAUUGUAUAAUAA B GACCUGAAUUGUAUAAUAArrB 710

R-008351922-000Z 991 210 GACCUGAAUUGUAUAAUAA UUAUUAUACAAUUCAGGUCUU 711

R-OO8352138-000K 860 37 AGUCCAUUGAUAUUUGGUC B AGUCCAUUGAUAUUUGGUC7TB 712

R-OO8352138-0O0K 860 37 AGUCCAUUGAUAUUUGGUC GACCAAAUAUCAAUGGACUUU 713

R-008351988-000F 577 61 UACAAGCUCUUGAAGACAC B UACAAGCUCUUGAAGAC AC JT B 714

R-008351988-000F 577 61 UACAAGCUCUUGAAGACAC GUGUCUUCAAGAGCUUGUAUU 715

R-O08352111-00OP 576 59 UUACAAGCUCUUGAAGACA B UUACAAGCUCUUGAAGACA rB 716

R-008352111-OOOP 576 59 UUACAAGCUCUUGAAGACA UGUCUUCAAGAGCUUGUAAUU 717

R-008352078-000V 535 64 AAAGAUGUAUAUAUAGUAC B A AAG AUGUAUAUAUAGUAC7T B 718

R-008352078-000V 535 64 AAAGAUGUAUAUAUAGUAC GUACUAUAUAUACAUCUUUUU 719

R-008351925-000A 1 177 88 CUGGAGCAGUAUUACGACC B CUGGAGCAGU AUUACGACC JT B 720

R-008351925-000A 1177 88 CUGGAGCA GU AUU ACG ACC GGUCGUAAUACUGCUCCAGUU 721

R-008351991-000M 832 40 AUUAUGUUGAAUUCCAAGG B AUUAUGUUGAAUUCCAAGG 7TB 722

R-008351991-000M 832 40 AUUAUGUUGAAUUCCAAGG CCUUGGAAUUCAACAUAAUUU 723

R-008351964-OOOL 468 56 GCGCUUCAGACAUGAGAAC B GCGCUUCAGAC AUGAGAACTT B 724

R-008351964-OOOL 468 56 GCGCUUCAGACAUGAGAAC GUUCUCAUGUCUGAAGCGCUU 725

R-008352141-OOOS 673 13 GUUCUGCACCGUGACCUCA B GUUCUGCACCGUGACCUCA7TB 726

R-0O8352141 -O00S 673 13 GUUCUGCACCGUGACCUCA UGAGGUCACGGUGCAGAACUU 727

R-008352117-OOOS 415 46 UUUGAGCACCAGACCUACU B UUUGAGCACCAGACCUACU7TB 728

R-008352117-OOOS 415 46 UUUGAGCACCAGACCUACU AGUAGGUCUGGUGCUCAAAUU 729

R-008352042-000R 474 51 CAGACAUGAGAACAUCAUU B CAG AC AUG AGA ACAUC AUU7T B 730

R-008352042-OOOR 474 51 CAGACAUGAGAACAUCAUU AAUGAUGUUCUCAUGUCUGUU 731

R-008352045-OOOS 771 105 CCAUGAUCACACAGGGUUC B CCAUGAUCACACAGGGUUCZTB 732

R-008352045-000S 771 105 CCAUGAUCACACAGGGUUC GAACCCUGUGUGAUCAUGGUU 733

R-O0835212O-000Y 1082 30 AUGCUGACUCCAAAGCUCU B AUGCUGACUCCAAAGCUCU 7TB 734 R-008352120-000Y 1082 30 AUGCUGACUCCAAAGCUCU AGAGCUUUGGAGUCAGCAUUU 735

R-008352048-OOOT 526 29 G AGC A A AUGAAAGAUGUA U B GAGCA A AUGAA AGAUGUAU 7 B 736

R-008352048-OOOT 526 29 GAGC A A AUG A A AGAUGUAU AUACAUCUUUCAUUUGCUCUU 737

R-008351928-000B 1 139 93 AGAGGAUUGAAGUAGAACA B AGAGGAUUGAAGUAGAACA 7 B 738

R-OO8351928-0O0B 1 139 93 AGAGGAUUGAAGUAGAACA UGUUCUACUUCAAUCCUCUUU 739

R-008352144-000T 482 1 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAA7TB 740

R-008352144-000T 482 1 AGAACAUCAUUGGAAUCAA UUGAUUCCAAUGAUGUUCUUU 741

R-008352147-000U 830 16 AAAUUAUGUUGAAUUCCAA B AAAUUAUGUUGAAUUCCAA7TB 742

R-008352147-OOOU 830 16 AAAUUAUGUUGAAUUCCAA UUGGAAUUCAACAUAAUUUUU 743

R-008351997-O0OP 1 140 1 15 GAGGAUUGAAGUAGAACAG B GAGGAUUGAAGUAGAACAG7TB 744

R-008351997-000P 1140 1 15 GAGGAUUGAAGUAGAACAG CUGUUCUACUUCAAUCCUCUU 745

R-008351967-00OM 1136 142 ACAAGAGGAUUGAAGUAGA B ACAAGAGGAUUGAAGUAGA 7TB 746

R-008351967-000M 1136 142 ACAAGAGGAUUGAAGUAGA UCUACUUCAAUCCUCUUGUUU 747

R-0O8352081-00OB 587 1 19 UGAAGACACAACACCUCAG B UGAAGACACAACACCUCAG7TB 748

R-008352081-OOOB 587 1 19 UGAAGACACAACACCUCAG CUGAGGUGUUGUGUCUUCAUU 749

R-008352024-000Y 1 146 122 UGAAGUAGAACAGGCUCUG B UGAAGUAGAACAGGCUCUG7TB 750

R-OO8352O24-0O0Y 1 146 122 UGAAGUAGAACAGGCUCUG CAGAGCCUGUUCUACUUCAUU 751

R-008352027-000Z 1076 25 UCCCAAAUGCUGACUCCAA B UCCCAAAUGCUGACUCCAA 7TB 752

R-O08352O27-OO0Z 1076 25 UCCCAAAUGCUGACUCCAA UUGGAGUCAGCAUUUGGGAUU 753

R-O08352084-000C 662 12 AUUCAGCUAACGUUCUGCA B AUUCAGCUAACGUUCUGCA7TB 754

R-008352084-000C 662 12 AUUCAGCUAACGUUCUGCA UGCAGAACGUUAGCUGAAUUU 755

R-008352123-000Z 357 154 GUGCUCUGCUUAUGAUAAU B GUGCUCUGCUUAUGAUAAU 7TB 756

R-008352123-OOOZ 357 154 GUGCUCUGCUUAUGAUAAU AUUAUCAUAAGCAGAGCACUU 757

R-008351931 -OOOH 460 60 AUCUUACUGCGCUUCAGAC B AUCUUACUGCGCUUCAGAC7rB 758

R-008351931-000H 460 60 AUCUUACUGCGCUUCAGAC GUCUGAAGCGCAGUAAGAUUU 759

R-008352087-OOOD 575 78 UUUACAAGCUCUUGAAGAC B UUUACAAGCUCUUGAAGAC7TB 760

R-008352087-OOOD 575 78 UUUACAAGCUCUUGAAGAC GUCUUCA AG AGCUUGU A A A UU 761

R-008352051-000Z 31 1 215 GCUACACCAACCUCUCGUA B GCUACACCAACCUCUCGUA7TB 762

R-008352051-000Z 31 1 215 GCUACACCAACCUCUCGUA UACGAGAGGUUGGUGUAGCUU 763

R-008352054-000A 759 100 UGCAGAUCCAGACCAUGAU B UGCAGAUCCAGACCAUGAU77B 764

R-008352054-OOOA 759 100 UGCAGAUCCAGACCAUGAU AUCAUGGUCUGGAUCUGCAUU 765

R-008352000-000D 314 216 ACACCAACCUCUCGUACAU B ACACCAACCUCUCGUACAU7TB 766

R-008352000-000D 314 216 ACACCAACCUCUCGUACAU AUGUACGAGAGGUUGGUGUUU 767

R-008352150-OOOA 664 79 UCAGCUAACGUUCUGCACC B UCAGCUA ACGUUCUGC ACC7T B 768

R-008352150-000A 664 79 UCAGCUAACGUUCUGCACC GGUGCAGAACGUUAGCUGAUU 769

R-008351934-000 J 661 23 CAUUCAGCUAACGUUCUGC B CAUUC AGCUAACGUUCUGC7T B 770

R-008351934-000J 661 23 CAUUCAGCUAACGUUCUGC GCAGAACGUUAGCUGAAUGUU 771

R-008352057-000B 856 70 ACCAAGUCCAUUGAUAUUU B ACCAAGUCCAUUGAUAUUU 7TB 772

R-008352057-000B 856 70 ACCAAGUCCAUUGAUAUUU AAAUAUCAAUGGACUUGGUUU 773

R-008352090-OOOK 658 62 AUCCAUUCAGCUAACGUUC B AUCC AUUCAGCU AACGUUC7T B 774

R-008352090-000K 658 62 AUCCAUUCAGCUAACGUUC GAACGUUAGCUGAAUGGAUUU 775

R-008352060-000H 992 21 1 ACCUGAAUUGUAUAAUAAA B ACCUGAAUUGUAUAAUAAA 2TB 776

R-OO835206O-OO0H 992 211 ACCUGAAUUGUAUAAUAAA UUUAUUAUACAAUUCAGGUUU 777

R-008352030-000F 1 138 102 AAGAGGAUUGAAGUAGAAC B AAGAGGAUUGAAGUAGAAC JTB 778

R-008352030-OOOF 1138 1 2 AAGAGGAUUGAAGUAGAAC GUUCU ACUUCA AUCCUCUUU U 779

R-008351937-000K 480 126 UGAGAACAUCAUUGGAAUC B UGAGAACAUCAUUGGAAUC7TB 780

R-008351937-000K 480 126 UGAGAACAUCAUUGGAAUC GAUUCCAAUGAUGUUCUCAUU 781

R-008351940-000S 467 55 UGCGCUUCAGACAUGAGAA B UGCGCUUCAGACAUGAGAA7TB 782

R-008351940-000S 467 55 UGCGCUUCAGACAUGAGAA UUCUCAUGUCUGAAGCGCAUU 783

R-008352063-000J 471 48 CUUCAGACAUGAGAACAUC B CUUC AGACAUGAGAACAUC7T B 784

R-008352063-OO J 471 48 CUUCAGACAUGAGAACAUC GAUGUUCUCAUGUCUGAAGUU 785 R-008352153-000B 470 50 GCUUCAGACAUGAGAACAU B GCUUCAGACAUGAGAACAU7TB 786

R-0O8352153-O00B 470 50 GCUUCAGACAUGAGAACAU AUGUUCUCAUGUCUGAAGCUU 787

R-008351970-OOOU 377 90 UCAACAAAGUUCGAGUAGC B UCAACAAAGUUCGAGUAGC 7TB 788

R-008351970-OOOU 377 90 UCAACAAAGUUCGAGUAGC GCUACUCGAACUUUGUUGAUU 789

R-008351973-000V 1052 18 AUAAGGUGCCAUGGAACAG B AUAAGGUGCCAUGGAACAG7TB 790

R-008351973-OOOV 1052 18 AUAAGGUGCCAUGGAACAG CUGUUCCAUGGCACCUUAUUU 791

R-008351976-000W 665 21 CAGCUAACGUUCUGCACCG B CAGCUA ACGUUCUGCACCG7T B 792

R-O08351976-0OOW 665 21 CAGCUAACGUUCUGCACCG CGGUGCAGAACGUUAGCUGUU 793

R-O08351943-0OOT 521 217 CCAUCGAGCAAAUGAAAGA B CCAUCGAGCAAAUGAAAGA 7TB 794

R-008351943-OOOT 521 217 CCAUCGAGCAAAUGAAAGA UCUUUCAUUUGCUCGAUGGUU 795

R-008352003-OOOE 518 171 CAACCAUCGAGCAAAUGAA B CAACCAUCGAGCAAAUGAA TT B 796

R-008352003-000E 518 171 CAACCAUCGAGCAAAUGAA UUCAUUUGCUCGAUGGUUGUU 797

R-008352066-OOOK 363 158 UGCUUAUGAUAAUGUCAAC B UGCUUAUGAUAAUGUCAAC7TB 798

R-OO8352O66-00OK 363 158 UGCUUAUGAUAAUGUCAAC GUUGACAUUAUCAUAAGCAUU 799

R-008352156-000C 864 97 CAUUGAUAUUUGGUCUGUA B CAUUGAUAUUUGGUCUGUA7TB 800

R-008352156-000C 864 97 CAUUGAUAUTJUGGUCUGUA UACAGACCAAAUAUCAAUGUU 801

R-OO8352OO6-000F 514 169 GCACCAACCAUCGAGCAAA B GCACCAACCAUCGAGCAAA7TB 802

R-008352006-000F 514 169 GCACCAACCAUCGAGCAAA UUUGCUCGAUGGUUGGUGCUU 803

R-008352159-000D 833 39 UUAUGUUGAAUUCCAAGGG B UUAUGUUGAAUUCCAAGGG 7TB 804

R-008352159-000D 833 39 UUAUGUUGAAUUCCAAGGG CCCUUGGAAUUCAACAUAAUU 805

R-008352069-OOOL 772 95 CAUGAUCACACAGGGUUCC B CAUGAUCACACAGGGUUCC 7TB 806

R-008352069-OOOL 772 95 CAUGAUCACACAGGGUUCC GGAACCCUGUGUGAUCAUGUU 807

R-008351946-000U 1170 81 CCCAUAUCUGGAGCAGUAU B CCCAUAUCUGGAGCAGUAU7TB 808

R-00835 I 46-OOOU 1 170 81 CCCAUAUCUGGAGCAGUAU AUACUGCUCCAGAUAUGGGUU 809

R-008351949-000V 765 111 UCCAGACCAUGAUCACACA B UCCAGACCAUGAUCACACA7TB 810

R-008351949-000V 765 1 11 UCCAGACCAUGAUCACACA UGUGUGAUCAUGGUCUGGAUU 81 1

R-008352033-000G 1078 72 CCAAAUGCUGACUCCAAAG B CCAAAUGCUGACUCCAAAG 7 B 812

R-008352033-OOOG 1078 72 CCAAAUGCUGACUCCAAAG CUUUGGAGUCAGCAUUUGGUU 813

R-O08352O36-00OH 536 52 AAGAUGUAUAUAUAGUACA B AAGAUGUAUAUAUAGUACA 7TB 814

R-008352036-OOOH 536 52 AAGAUGUAUAUAUAGUACA UGUACUAUAUAUACAUCUUUU 815

R-O08352O72-0O0T 533 66 UGAAAGAUGU AUAUAUAGU B UGAAAGAUGUAUAUAUAGU TTB 816

R-008352072-000T 533 66 UGAAAGAUGUAUAUAUAGU ACUAUAUAUACAUCUUUCAUU 817

R-008352162-000K 465 57 ACUGCGCUUCAGACAUGAG B ACUGCGCUUCAGACAUGAG TT B 818

R-008352162-000K 465 57 ACUGCGCUUCAGACAUGAG CUCAUGUCUGAAGCGCAGUUU 819

R-008352129-000B 681 31 CCGUGACCUCAAGCCUUCC B CCGUGACCUCAAGCCUUCC7 B 820

R-008352129-OOOB 681 31 CCGUGACCUCAAGCCUUCC GGAAGGCUUGAGGUCACGGUU 821

R-008352039-000J 688 108 CUCAAGCCUUCCAACCUGC B CUCAAGCCUUCC AACCUGC7T B 822

R-008352039-000J 688 108 CUCAAGCCUUCCAACCUGC GCAGGUUGGAAGGCUUGAGUU 823

R-008351979-000X 863 9 CCAUUGAUAUUUGGUCUGU B CCAUUGAUAUUUGGUCUGU 7 B 824

R-008351979-000X 863 9 CCAUUGAUAUUUGGUCUGU ACAGACCAAAUAUCAAUGGUU 825

R-008352093-OOOL 586 127 UUGAAGACACAACACCUCA B UUGAAGACACAACACCUCA 7TB 826

R-008352093-OOOL 586 127 UUGAAGACACAACACCUCA UGAGGUGUUGUGUCUUCAAUU 827

R-008351952-000B 666 5 AGCUAACGUUCUGCACCGU B AGCUA ACGUUCUGCACCGU7T B 828

R-008351952-OOOB 666 5 AGCUAACGUUCUGCACCGU ACGGUGCAGAACGUUAGCUUU 829

R-008351955-0O0C 831 69 AAUUAUGUUGAAUUCCAAG B AAUUAUGUUGAAUUCCAAG7TB 830

R-008351955-000C 831 69 AAUUAUGUUGAAUUCCAAG CUUGGAAUUCAACAUAAUUUU 831

R-008352009-000G 478 80 CAUGAGAACAUCAUUGGAA B CAUGAGAACAUCAUUGGAA JTB 832

R-008352009-000G 478 80 CAUGAGAACAUCAUUGGAA UUCCAAUGAUGUUCUCAUGUU 833

R-008351982-000D 668 77 CUAACGUUCUGCACCGUGA B CUAACGUUCUGCACCGUGA 7TB 834

R-008351982-OOOD 668 77 CUAACGUUCUGCACCGUGA UCACGGUGCAGAACGUUAGUU 835

R-O08352165-0OOL 1081 19 AAUGCUGACUCCAAAGCUC B AAUGCUGACUCCAAAGCUC7TB 836 R-008352165-OOOL 1081 19 AAUGCUGACUCCAAAGCUC GAGCUUUGGAGUCAGCAUUUU 837

R-008352132-OOOH 1075 68 UUCCCAAAUGCUGACUCCA B UUCCCAAAUGCUGACUCCA 7T B 838

R-0O8352132-000H 1075 68 UUCCCAAAUGCUGACUCCA UGGAGUCAGCAUUUGGGAAUU 839

R-0O8352096-O00M 534 67 GAAAGAUGUAUAUAUAGUA B GAAAGAUGUAUAUAUAGUA 7TB 840

R-008352096-OOOM 534 67 GAAAGAUGUAUAUAUAGUA UACUAUAUAUACAUCUUUCUU 841

R-008352075-OOOU 1137 109 CAAGAGGAUUGAAGUAGAA B CAAGAGGAUUGAAGUAGAA7TB 842

R-008352075-000U 1 137 109 CAAGAGGAUUGAAGUAGAA UUCU ACUUCA AUCCUCUUGUU 843

R-008352168-OOOM 869 92 AUAUUUGGUCUGUAGGCUG B AUAUUUGGUCUGUAGGCUGrrB 844

R-008352168-000M 869 92 AUAUUUGGUCUGUAGGCUG CAGCCUACAGACCAAAUAUUU 845

R-008404887-000D 1018 129 GCUAGGAACUAUUUGCUUU B GCUAGGAACUAUUUGCUUU2 B 846

R-008404887-000D 1018 129 GCUAGGAACUAUUUGCUUU AAAGCAAAUAGUUCCUAGCUU 847

R-008404809-000G 1031 218 UGCUUUCUCUUCCACACAA B UGCUUUCUCUUCCACACAArrB 848

R-008404809-000G 1031 218 UGCUUUCUCUUCCACACAA UUGUGUGGAAGAGAAAGCAUU 849

R-008404659-000Z 1070 133 GGCUGUUCCCAAAUGCUGA B GGCUGUUCCCA A AUGCUG ATT B 850

R-008404659-000Z 1070 133 GGCUGUUCCCAAAUGCUGA UCAGCAUUUGGGAACAGCCUU 851

R-O08404815-000P 1088 128 ACUCCAAAGCUCUGGACUU B ACUCCAAAGCUCUGGACUU 7TB 852

R-008404815-OOOP 1088 128 ACUCCAAAGCUCUGGACUU AAGUCCAGAGCUUUGGAGUUU 853

R-008404710-000L 1097 136 CUCUGGACUUAUUGGACAA B CUCUGG ACUUAUUGG AC A ATT B 854

R-008404710-000L 1097 136 CUCUGGACUUAUUGGACAA UUGUCCAAUAAGUCCAGAGUU 855

R-008404779-OOOU 1098 137 UCUGGACUUAUUGGACAAA B UCUGGACUUAUUGGACAAA 2 B 856

R-008404779-OOOU 1098 137 UCUGGACUUAUUGGACAAA UUUGUCCAAUAAGUCCAGAUU 857

R-008404767-OOOJ 1118 138 UGUUGACAUUCAACCCACA B UGUUGACAUUCAACCCACA7TB 858

R-008404767-OOOJ 1 1 18 138 UGUUGACAUUCAACCCACA UGUGGGUUGAAUGUCAACAUU 859

R-008404677-000S 1 120 219 UUGACAUUCAACCCACACA B UUGACAUUCAACCCACACA2TB 860

R-008404677-OOOS 1 120 219 UUGACAUUCAACCCACACA UGUGUGGGUUGAAUGUCAAUU 861

R-008404833-OOOG 1124 220 CAUUCAACCCACACAAGAG B CAUUCA ACCC ACAC A AG AG2T B 862

R-008404833-OOOG 1 124 220 CAUUCAACCCACACAAGAG CUCUUGUGUGGGUUGAAUGUU 863

R-O084O4824-O00Y 1 133 141 CACACAAGAGGAUUGAAGU B CACACAAGAGGAUUGAAGU 2TB 864

R-008404824-OOOY 1133 141 CACACAAGAGGAUUGAAGU ACUUCAAUCCUCUUGUGUGUU 865

R-O08404665-O00G 1143 221 GAUUGAAGUAGAACAGGCU B GAUUGAAGUAGAACAGGCUr/'B 866

R-008404665-OOOG 1 143 221 GAUUGAAGUAGAACAGGCU AGCCUGUUCUACUUCAAUCUU 867

R-O08404857-OO0B 1164 222 GGCCCACCCAUAUCUGGAG B GGCCCACCCAUAUCUGGAG TT B 868

R-008404857-000B 1 164 222 GGCCCACCCAUAUCUGGAG CUCCAGAUAUGGGUGGGCCUU 869

R-008404818-OOOR 1 168 143 CACCCAUAUCUGGAGCAGU B CACCCAUAUCUGGAGCAGUrrB 870

R-008404818-000R 1 168 143 CACCCAUAUCUGGAGCAGU ACUGCUCCAGAUAUGGGUGUU 871

R-008404869-000L 1200 223 UGACGAGCCCAUCGCCGAA B UG ACGAGCCCAUCGCCGA ATT B 872

R-008404869-OOOL 1200 223 UGACGAGCCCAUCGCCGAA UUCGGCGAUGGGCUCGUCAUU 873

R-008404701 -000C 1212 147 CGCCGAAGCACCAUUCAAG B CGCCGAAGCACCAUUCA AG rr B 874

R-008404701-OOOC 1212 147 CGCCGAAGCACCAUUCAAG CUUGAAUGGUGCUUCGGCGUU 875

R-008404893-000L 1244 148 UGG AUGACUUGCCU A AG G A B UGGAUGACUUGCCUAAGGA 2 B 876

R-008404893-OOOL 1244 148 UGGAUGACUUGCCUAAGGA UCCUUAGGCAAGUCAUCCAUU 877

R-008404782-OOOA 1245 149 GGAUGACUUGCCUAAGGAA B GGAUGACUUGCCUAAGGAA B 878

R-0O8404782-000A 1245 149 GGAUGACUUGCCUAAGGAA UUCCUUAGGCAAGUCAUCCUU 879

R-008404713-OOOM 1284 6 UGAAGAGACUGCUAGAUUC B UGAAGAGACUGCUAGAUUC2TB 880

R-OO8404713-000 1284 6 UGAAGAGACUGCUAGAUUC GAAUCUAGCAGUCUCUUCAUU 881

R-008404680-OOOY 1294 20 GCUAGAUUCCAGCCAGGAU B GCUAGAUUCCAGCCAGGAU7TB 882

R-008404680-OOOY 1294 20 GCUAGAUUCCAGCCAGGAU AUCCUGGCUGGAAUCUAGCUU 883

R-008404890-OOOK 1295 28 CUAGAUUCCAGCCAGGAUA B CUAGAUUCCAGCCAGGAUA2TB 884

R-008404890-OOOK 1295 28 CUAGAUUCCAGCCAGGAUA UAUCCUGGCUGGAAUCUAGUU 885

R-008404827-OOOZ 1303 32 CAGCCAGGAUACAGAUCUU B CAGCCAGGAUAC AGAUCUU7T B 886

R-008404827-OOOZ 1303 32 CAGCCAGGAUACAGAUCUU AAGAUCUGUAUCCUGGCUGUU 887 R-008404839-OOOJ 353 224 UGGUGUGCUCUGCUUAUGA B UGGUGUGCUCUGCUUAUGA 7TB 888

R-008404839-OOOJ 353 224 UGGUGUGCUCUGCUUAUGA UCAUAAGCAGAGCACACCAU 889

R-008404872-OOOT 355 225 GUGUGCUCUGCUUAUGAUA B GUGUGCUCUGCUUAUGAUA7TB 890

R-008404872-OOOT 355 225 GUGUGCUCUGCUUAUGAUA UAUCAUAAGCAGAGCACACUU 891

R-008404743-OOOP 358 155 UGCUCUGCUUAUGAUAAUG B UGCUCUGCUUAUGAUAAUG7TB 892

R-008404743-000P 358 155 UGCUCUGCUUAUGAUAAUG CAUUAUCAUAAGCAGAGCAUU 893

R-008404668-OOOH 362 226 CUGCUUAUGAUAAUGUCAA B CUGCUUAUGAUAAUGUCAA TT B 894

R-008404668-OOOH 362 226 CUGCUUAUGAUAAUGUCAA UUGACAUUAUCAUAAGCAGUU 895

R-008404773-OOOS 368 159 AUGAUAAUGUCAACAAAGU B AUGAUAAUGUCAACAAAGUTTB 896

R-008404773-OO S 368 159 AUGAUAAUGUCAACAAAGU ACUUUGUUGACAUUAUCAUUU 897

R-008404731-000E 437 161 AGAGAACCCUGAGGGAGAU B AGAGAACCCUGAGGGAGAU7TB 898

R-008404731-OOOE 437 161 AGAGAACCCUGAGGGAGAU AUCUCCCUCAGGGUUCUCUUU 899

R-008404653-OOOX 477 227 ACAUGAGAACAUCAUUGGA B ACAUGAGAACAUCAUUGGA TB 900

R-008404653-OOOX 477 227 ACAUGAGAACAUCAUUGGA UCCAAUGAUGUUCUCAUGUUU 901

R-008404812-OOON 489 228 CAUUGGAAUCAAUGACAUU B CAUUGGAAUCAAUGACAUU 7 B 902

R-008404812-000N 489 228 CAUUGGAAUCAAUGACAUU AAUGUCAUUGAUUCCAAUGUU 903

R-008404725-O0OX 490 163 AUUGGAAUCAAUGACAUUA B AUUGGAAUCAAUGACAUUA7TB 904

R-008404725-000X 490 163 AUUGGAAUCAAUGACAUUA UAAUGUCAUUGAUUCCAAUUU 905

R-008404755-000Z 491 164 UUGGAAUCAAUGACAUUAU B UUGGAAUCAAUGACAUUAU7TB 906

R-008404755-OOOZ 491 164 UUGGAAUCAAUGACAUUAU AUAAUGUCAUUGAUUCCAAUU 907

R-008404746-000R 492 165 UGGAAUCAAUGACAUUAUU B UGGAAUCAAUGACAUUAUUTTB 908

R-008404746-000R 492 165 UGGAAUCAAUGACAUUAUU AAUAAUGUCAUUGAUUCCAUU 909

R-008404689-000B 494 229 GAAUCAAUGACAUUAUUCG B GAAUCAAUGACAUUAUUCG 7TB 910

R-008404689-000B 494 229 GAAUCAAUGACAUUAUUCG CGAAUAAUGUCAUUGAUUCUU 911

R-008404836-000H 495 166 AAUCAAUGACAUUAUUCGA B AAUCAAUGACAUUAUUCGA 7T B 912

R-008404836-000H 495 166 A AUCA AUG ACAU UAUUCG A UCGAAUAAUGUCAUUGAUUUU 913

R-008404719-000P 497 230 UCAAUGACAUUAUUCGAGC B UCAAUGACAUUAUUCGAGC7TB 914

R-008404719-000P 497 230 UCAAUGACAUUAUUCGAGC GCUCGAAUAAUGUCAUUGAUU 915

R-008404800-000D 498 167 CAAUGACAUUAUUCGAGCA B CAAUGACAUUAUUCGAGCA7TB 916

R-008404800-OOOD 498 167 CAAUGACAUUAUUCGAGCA UGCUCGAAUAAUGUCAUUGUU 917

R-008404707-000E 503 231 ACAUUAUUCGAGCACCAAC B AC AUU AUUCGAGCACCA AC7T B 918

R-008404707-000E 503 23 ACAUUAUUCGAGCACCAAC GUUGGUGCUCGAAUAAUGUUU 919

R-008404821-OOOX 515 170 CACCAACCAUCGAGCAAAU B CACCAACCAUCGAGCAAAU 7T B 920

R-008404821-OO X 515 170 CACCAACCAUCGAGCAAAU AUUUGCUCGAUGGUUGGUGUU 921

R-O08404683-O00Z 522 84 CAUCGAGCAAAUGAAAGAU B CAUCGAGCAAAUGAAAGAU Γ B 922

R-008404683-OOOZ 522 84 CAUCGAGCAAAUGAAAGAU AUCUUUCAUUUGCUCGAUGUU 923

R-008404686-000A 525 174 CGAGCAAAUGAAAGAUGUA B CGAGCAAAUGAAAGAUGUA7TB 924

R-008404686-O00A 525 174 CGAGCAAAUGAAAGAUGUA UACAUCUUUCAUUUGCUCGUU 925

R-008404737-OOOG 528 232 GCAAAUGAAAGAUGUAUAU B GC AA AUGA AAGAUGUAUAU7T B 926

R-008404737-OOOG 528 232 GCAAAUGAAAGAUGUAUAU AUAUACAUCUUUCAUUUGCUU 927

R-008404854-OOOA 538 175 GAUGUAUAUAUAGUACAGG B GAUGUAUAUAUAGUACAGG7TB 928

R-008404854-OOOA 538 175 GAUGUAUAUAUAGUACAGG CCUGUACUAUAUAUACAUCUU 929

R-008404662-OOOF 540 176 UGUAUAUAUAGUACAGGAC B UGUAUAUAU AGUACAGGAC7T B 930

R-008404662-000F 540 176 UGUAUAUAUAGUACAGGAC GUCCUGUACUAUAUAUACAUU 931

R-008404704-000D 542 177 UAUAUAUAGUACAGGACCU B UAUAUAUAGUACAGGACCU7TB 932

R-008404704-000D 542 177 UAUAUAUAGUACAGGACCU AGGUCCUGUACUAUAUAUAUU 933

R-008404764-000H 549 233 AGUACAGGACCUCAUGGAA B AGUACAGGACCUCAUGGAA 7TB 934

R-008404764-000H 549 233 AGUACAGGACCUCAUGGAA UUCCAUGAGGUCCUGUACUUU 935

R-008404698-000K 551 178 UACAGGACCUCAUGGAAAC B UACAGGACCUCAUGGAAAC TT B 936

R-008404698-000K 551 178 UACAGGACCUCAUGGAAAC GUUUCCAUGAGGUCCUGUAUU 937

R-008404734-000F 557 234 ACCUCAUGGAAACAGAUCU B ACCUCAUGGAA AC AG AUCU7T B 938 R-008404734-000F 557 234 ACCUCAUGGAAACAGAUCU AGAUCUGUUUCCAUGAGGUUU 939

R-008404875-000U 563 235 UGGAAACAGAUCUUUACAA B UGGAAACAGAUCUUUACAA 7TB 940

R-008404875-000U 563 235 UGGAAACAGAUCUUUACAA UUGUAAAGAUCUGUUUCCAUU 941

R-008404749-000S 564 236 GGAAACAGAUCUUUACAAG B GGAAACAGAUCUUUACAAG7TB 942

R-008404749-000S 564 236 GGAAACAGAUCUUUACAAG CUUGUAAAGAUCUGUUUCCUU 943

R-008404656-OOOY 565 237 GAAACAGAUCUUUACAAGC B G A AAC AG AUCUUUACAAGC7T B 944

R-008404656-000Y 565 237 GAAACAGAUCUUUACAAGC GCUUGUAAAGAUCUGUUUCUU 945

R-008404851-000Z 567 182 AACAGAUCUUUACAAGCUC B AACAGAUCUUU ACA AGCUC2T B 946

R-008404851-OOOZ 567 182 AACAGAUCUUUACAAGCUC GAGCUUGUAAAGAUCUGUUUU 947

R-008404716-OOON 572 110 AUCUUUACAAGCUCUUGAA B AUCUU ACAAGCUCUUGAA7TB 948

R-008404716-000N 572 110 AUCUTJUACAAGCUCUUGAA UUCAAGAGCUUGUAAAGAUUU 949

R-008404788-OOOC 584 238 UCUUGAAGACACAACACCU B UCUUG A AG AC AC A AC ACCU7T B 950

R-008404788-OOOC 584 238 UCUUGAAGACACAACACCU AGGUGUUGUGUCUUCAAGAUU 951

R-008404770-OOOR 594 185 ACAACACCUCAGCAAUGAC B AC A ACACCUC AGCA AUGAC7T B 952

R-008404770-000R 594 185 ACAACACCUCAGCAAUGAC GUCAUUGCUGAGGUGUUGUUU 953

R-00840483O-0OOF 599 239 ACCUCAGCAAUGACCAUAU B ACCUCAGCAAUGACCAUAU7TB 954

R-008404830-OOOF 599 239 ACCUCAGCAAUGACCAUAU AUAUGGUCAUUGCUGAGGUUU 955

R-008404674-000R 632 186 ACCAGAUCCUCAGAGGGUU B ACCAGAUCCUCAGAGGGUU 7TB 956

R-008404674-000R 632 186 ACCAGAUCCUCAGAGGGUU AACCCUCUGAGGAUCUGGUUU 957

R-008404896-000M 687 240 CCUCAAGCCUUCCAACCUG B CCUC AAGCCUUCC A ACCUG7T B 958

R-008404896-000M 687 240 CCUCAAGCCUUCCAACCUG CAGGUUGGAAGGCUUGAGGUU 959

R-008404791 -000J 695 241 CUUCCAACCUGCUGCUCAA B CUUCCAACCUGCUGCUCAA 7 B 960

R-008404791-000J 695 241 CUUCCAACCUGCUGCUCAA UUGAGCAGCAGGUUGGAAGUU 961

R-008404794-000K 707 242 UGCUCAACACCACCUGUGA B UGCUCAACACCACCUGUGATTB 962

R-008404794-OOO 707 242 UGCUCAACACCACCUGUGA UCACAGGUGGUGUUGAGCAUU 963

R-008404740-OOON 713 243 ACACCACCUGUGAUCUCAA B ACACCACCUGUGAUCUCAATTB 964

R-008404740-OOON 713 243 ACACCACCUGUGAUCUCAA UUGAGAUCACAGGUGGUGUUU 965

R-008404761-OOOG 720 190 CUGUGAUCUCAAGAUCUGU B CUGUGAUCUCAAGAUCUGU 7TB 966

R-008404761-OOOG 720 190 CUGUGAUCUCAAGAUCUGU ACAGAUCUUGAGAUCACAGUU 967

R-008404845-OOOS 725 191 AUCUCAAGAUCUGUGACUU B AUCUCAAGAUCUGUGACUU2TB 968

R-008404845-OOOS 725 191 AUCUCAAGAUCUGUGACUU AAGUCACAGAUCUUGAGAUUU 969

R-008404797-OOOL 753 244 CCGUGUUGCAGAUCCAGAC B CCGUGUUGCAGAUCCAGAC7TB 970

R-O08404797-000L 753 244 CCGUGUUGCAGAUCCAGAC GUCUGGAUCUGCAACACGGUU 971

R-008404776-OOOT 778 245 CACACAGGGUUCCUGACAG B CACACAGGGUUCCUGACAG ΓΒ 972

R-008404776-OOOT 778 245 CACACAGGGUUCCUGACAG CUGUCAGGAACCCUGUGUGUU 973

R-008404881-000B 782 194 CAGGGUUCCUGACAGAAUA B CAGGGUUCCUGAC AGAAUA7T B 974

R-008404881-0O0B 782 194 CAGGGUUCCUGACAGAAUA UAUUCUGUCAGGAACCCUGUU 975

R-008404671-OO0P 823 26 GCUCCAGAAAUUAUGUUGA B GCUCCAGAAAUUAUGUUGA7TB 976

R-008404671-000P 823 26 GCUCCAGAAAUUAUGUUGA UCAACAUAAUUUCUGGAGCUU 977

R-008404878-OOOV 825 34 UCCAGAAAUUAUGUUGAAU B UCCAGAAAUUAUGUUGAAU7TB 978

R-008404878-OOOV 825 34 UCCAGAAAUUAUGUUGAAU AUUCAACAUAAUUUCUGGAUU 979

R-OO8404758-0O0A 826 24 CCAGAAAUUAUGUUGAAUU B CCAGAAAUUAUGUUGAAUU 7TB 980

R-008404758-OOOA 826 24 CCAGAAAUUAUGUUGAAUU AAUUCAACAUAAUUUCUGGUU 981

R-008404848-000T 829 198 GAAAUUAUGUUGAAUUCCA B GAAAUUAUGUUGAAUUCCA 7TB 982

R-008404848-000T 829 198 GAAAUUAUGUUGAAUUCCA UGGAAUUCAACAUAAUUUCUU 983

R-008404863-000J 849 246 GGGCUACACCAAGUCCAUU B GGGCUACACCAAGUCCAUU TT B 984

R-008404863-000J 849 246 GGGCUACACCAAGUCCAUU AAUGGACUUGGUGUAGCCCUU 985

R-008404899-000N 854 82 ACACCAAGUCCAUUGAUAU B ACACCAAGUCCAUUGAUAU7TB 986

R-008404899-000N 854 82 ACACCAAGUCCAUUGAUAU AUAUCAAUGGACUUGGUGUUU 987

R-008404902-000F 855 106 CACCAAGUCCAUUGAUAUU B CACCAAGUCCAUUGAUAUU 7T B 988

R-008404902-000F 855 106 CACCAAGUCCAUUGAUAUU AAUAUCAAUGGACUUGGUGUU 989

R-008382273-000N 366 4 UUAUGAUAAUGUCAACAAA UUUGuuGAcAuuAucAuAAUsU 1017

R-008382230-000T 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1029

R-008382230-OOOT 366 4 UUAUGAUAAUGUCAACAAA UUUGUUGACAUUAUCAUAAUSU 1030

R-008382193-000N 366 4 UUAUGAUAAUGUCAACAAA UUUGuuGAcAuuAucAuAAUsU 1014

R-008382193-000N 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382282-OOOX 366 4 UUAUGAUAAUGUCAACAAA B U AUGAUAAUGUCAACAAAUSU B 1031

R-008382282-OOOX 366 4 UUAUGAUAAUGUCAACAAA UUUGuuGAcAuuAucAuAAUsU 1017

R-00838225I-0OOL 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382251-000L 366 4 UUAUGAUAAUGUCAACAAA UUUgUUgaCaUUaUCaUaaUsU 1018

R-008382250-000C 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382250-000C 366 4 UUAUGAUAAUGUCAACAAA UUUGUUGACAUUAUCAUAAUSU 1019

R-008382232-000K 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382232-000K 366 4 UUAUGAUAAUGUCAACAAA UUUGUUGACAUUAUCAUAAUSU 1020

R-008382226-OOOC 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382226-000C 366 4 UUAUGAUAAUGUCAACAAA UUUgUUgaCaUUaUCaUaaUsU 1021

R-008382507-OOOF 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382507-OOOF 366 4 UUAUGAUAAUGUCAACAAA UUuGUUGACaUUAuCAuaaUsU 1032

R-008382532-OOOP 366 4 UUAUGAUAAUGUCAACAAA uuugUugACAUuaUCAUAaUsU 1033

R-008382S32-000P 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382549-000T 366 4 UUAUGAUAAUGUCAACAAA uUUGuUgacAuUauCAUAAUsU 1034

R-008382549-000T 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382556-000J 366 4 UUAUGAUAAUGUCAACAAA uUuGUUeacAUUAucaUAAUsU 1035

R-008382556-000J 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382566-OOOB 366 4 UUAUGAUAAUGUCAACAAA uUuGUUGACAUUAuCauaaUsU 1036

R-008382566-000B 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382578-00OL 366 4 UUAUGAUAAUGUCAACAAA uuuGUUsACaUUAuCAUAAUsU 1037

R-O08382578-O0OL 366 4 UUAUGAUAAUGUCAACAAA B UUAUGAUAAUGUCAACAAAUSU B 1031

R-008382283-000F 366 4 UUAUGAUAAUGUCAACAAA UUUGuuGAcAuuAucAuAAUsU 1014

R-008382283-000F 366 4 UUAUGAUAAUGUCAACAAA B UuAuGAuAAuGucAAcAAAUsU B 1038

R-008382157-000D 366 4 UUAUGAUAAUGUCAACAAA B uuAuGAuAAuGucAAcAAAUsU B 1038

R-008382157-000D 366 4 UUAUGAUAAUGUCAACAAA UUUGuuGAcAuuAucAuAAUsU 1017

R-008382227-OOOL 366 4 UUAUGAUAAUGUCAACAAA B UuAuGAuAAuGucAAcAAAUsU B 1038

R-008382227-000L 366 4 UUAUGAUAAUGUCAACAAA UUUgUUgaCaUUaUCaUaaUsU 1018

R-008382262-000M 366 4 UUAUGAUAAUGUCAACAAA B UuAuGAuAAuGucAAcAAAUsU B 1038

R-008382262-000M 366 4 UUAUGAUAAUGUCAACAAA UUUGUUGACAUUAUCAUAAUSU 1019

R-008382268-000P 366 4 UUAUGAUAAUGUCAACAAA B uuAuGAuAAuGucAAcAAAUsU B 1038

R-008382268-OOOP 366 4 UUAUGAUAAUGUCAACAAA UUUGUUGACAUUAUCAUAAUSU 1020

R-008382270-000M 366 4 UUAUGAUAAUGUCAACAAA B uAuGAuAAuGucAAcAAAUsU B 1038

R-008382270-OOOM 366 4 UUAUGAUAAUGUCAACAAA UUUgUUgaCaUUaUCaUaaUsU 1021

R-008382530-OOOX 366 4 UUAUGAUAAUGUCAACAAA B uuAuGAuAAuGucAAcAAAUsU B 1038

R-008382530-000X 366 4 UUAUGAUAAUGUCAACAAA UUUGUuGaCauUaucaUAAUsU 1039

R-008382531-000F 366 4 UUAUGAUAAUGUCAACAAA B UuAuGAuAAuGucAAcAAAUsU B 1038

R-008382531-000F 366 4 UUAUGAUAAUGUCAACAAA UUUGUugacaUUAucAUaaUsU 1040

R-008382539-000 A 366 4 UUAUGAUAAUGUCAACAAA B UuAuGAuAAuGucAAcAAAUsU B 1038

R-008382539-000A 366 4 UUAUGAUAAUGUCAACAAA UuU^uuGACaUUAUcaUAaUsU 1041

R-008382540-000P 366 4 UUAUGAUAAUGUCAACAAA uUUGUuGaCAuuaUCAUaaUsU 1042

R-008382540-000P 366 4 UUAUGAUAAUGUCAACAAA B UuAuGAuAAuGucAAcAAAUsU B 1038

R-008382550-000G 366 4 UUAUGAUAAUGUCAACAAA uUusUUGACAuUAucaUaaUsU 1043

R-008382550-000G 366 4 UUAUGAUAAUGUCAACAAA B uAuGAuAAuGucAAcAAAUsU B 1038

R-008382577-000C 366 4 UUAUGAUAAUGUCAACAAA B UuAuGAuAAuGucAAcAAAUsU B 1038

R-008382577-000C 366 4 UUAUGAUAAUGUCAACAAA UuUgUUgAcAUUauCAuaAUsU 1044

R-OOS382263-O00W 482 1 AGAACAUCAUUGGAAUCAA uuGAuuccAAuGAuGuucuUsU 1063

R-0O8382263-O0OW 482 1 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382265-OOON 482 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382265-OOON 482 AGAACAUCAUUGGAAUCAA UUGAuuccAAuGAuGuucuUsU 1066

R-008382264-000E 482 1 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382264-OOOE 482 1 AGAACAUCAUUGGAAUCAA UUGaUUCCaaUaaUeUUCUUsU 1067

R-00S382176-000E 482 1 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382176-000E 482 AGAACAUCAUUGGAAUCAA UUGAUUCCAAUGAUGUUCUUsU 1068

R-008382239-OOOW 482 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382239-000W 482 AGAACAUCAUUGGAAUCAA UUGAUUCCAAUGAUGUUCUUsU 1069

R-008382221-OOOJ 482 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382221-OOOJ 482 AGAACAUCAUUGGAAUCAA UUGaUUCCaaUgaUgUUCUUsU 1070

R-O08382514-OO0X 482 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-O08382514-OO0X 482 AGAACAUCAUUGGAAUCAA UUgAUUcCaAUGAugUUcUUsU 1081

R-008382515-OOOF 482 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-OO8382515-0O0F 482 AGAACAUCAUUGGAAUCAA UUGAUUCCaAUGAugUUcuUsU 1082

R-00838252S-OOOZ 482 1 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382528-OOOZ 482 AGAACAUCAUUGGAAUCAA UUgaUUCcAaueAuGuUCUUsU 1083

R-008382544-000Z 482 I AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382544-000Z 482 1 AGAACAUCAUUGGAAUCAA UUGAUuCcAaUGaUguUcuUsU 1084

R-008382570-000S 482 1 AGAACAUCAUUGGAAUCAA uUgAuUCcaAUgAUGuuCUUsU 1085

R-OO8382570-000S 482 1 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382575-000K 482 AGAACAUCAUUGGAAUCAA B AGAACAUCAUUGGAAUCAAUsU B 1080

R-008382575-000K 482 AGAACAUCAUUGGAAUCAA UuGaUuCCAAUgaUGUUcuUsU 1086

R-0O8382169-000N 482 1 AGAACAUCAUUGGAAUCAA uuGAuuccAAuGAuGuucuUsU 1063

R-008382169-000N 482 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-008382186-000X 482 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-OO8382186-000X 482 j AGAACAUCAUUGGAAUCAA UUGAuuccAAuGAuGuucuUsU 1066

R-008382259-00OF 482 1 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-008382259-OOOF 482 1 AGAACAUCAUUGGAAUCAA UUGaUUCCaaUeaUeUUCUUsU 1067

R-008382190-OOOM 482 1 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-008382190-OOOM 482 1 AGAACAUCAUUGGAAUCAA UUGAUUCCAAUGAUGUUCUUsU 1068

R-008382238-OOOM 482 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-OO8382238-0O0M 482 1 AGAACAUCAUUGGAAUCAA UUGAUUCCAAUGAUGUUCUUsU 1069

R-008382181-000D 482 1 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-008382181-000D 482 1 AGAACAUCAUUGGAAUCAA UUGaUUCCaaUgaUgUUCUUsU 1070

R-OO8382516-000P 482 1 AGAACAUCAUUGGAAUCAA uuGAUUCcAaUGAugUUCuUsU 1088

R-008382516-000P 482 1 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-008382527-000R 482 1 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-OO8382527-000R 482 1 AGAACAUCAUUGGAAUCAA UIJgAUuCCaAuGaUGUuCUUsU 1089

R-008382543-000R 482 1 AGAACAUCAUUGGAAUCAA uuGAUUCCAaUgAuGUuCUUsU 1090

R-008382543-OOOR 482 1 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-008382547-000 A 482 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-008382547-000 A 482 AGAACAUCAUUGGAAUCAA UUGauuCCAaUGauguuCUUsU 1091

R-0083825 1-OOOR 482 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-OO8382551-O00R 482 AGAACAUCAUUGGAAUCAA UUGAUUCcaAUaAuGuUCUUsU 1092

R-008382573-OOOT 482 AGAACAUCAUUGGAAUCAA B AGAAcAucAuuGGAAucAAUsU B 1087

R-0O8382573-O0OT 482 AGAACAUCAUUGGAAUCAA UUgaUUcCAAU_gAugUUcuUsU 1093

R-0O8382622-00OG 482 AGAACAUCAUUGGAAUCAA UuGAuuccAAuGAuGuucuUsU 1063

R-008382622-000G 482 AGAACAUCAUUGGAAUCAA B AGAACAVCAUUggaAuCsLAOsU B 1094

R-008382623-000R 482 AGAACAUCAUUGGAAUCAA UuGAuuccAAuGAuGuucuUsU 3063

R-008382244-000 V 657 2 UAUCCAUUCAGCUAACGUU AACGuuAGcuGAAuGGAuAUsU 1 115

R-008382163-OOOL 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1129

R-OO8382163-000L 657 2 UAUCCAUUCAGCUAACGUU AACgUUagCUgaaUggaUaUsU 11 16

R-008382257-000N 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1 129

R-008382257-000N 657 2 UAUCCAUUCAGCUAACGUU AACGUUAGCUGAAUGGAUAUsU 1 1 17

R-00838227 l-OOOW 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1129

R-OO8382271-O00W 657 2 UAUCCAUUCAGCUAACGUU AACGUUAGCUGAAUGGAUAUsU 1 1 18

R-008382187-000F 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1 129

R-008382187-000F 657 2 UAUCCAUUCAGCUAACGUU AACgUUagCUgaaUggaUaUsU 1119

R-008382536- OOZ 657 2 UAUCCAUUCAGCUAACGUU aaC^uUagcUgAAUgGAuAUsU 1 130

R-008382536-OOOZ 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1 129

R-008382542-OOOG 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1129

R-O08382542-0O0G 657 2 UAUCCAUUCAGCUAACGUU AacGUuaGCUGAAuGGaUaUsU 1 131

R-008382546-000S 657 2 UAUCCAUUCAGCUAACGUU aaCGuuAGCugAAUgGaUAUsU 1 132

R-008382546-000S 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1 129

R-008382561-000H 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1 129

R-008382561-OOOH 657 2 UAUCCAUUCAGCUAACGUU AACGuUAGcugAaUggAUAUsU 1 133

R-008382562-OOOS 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1129

R-008382562-000S 657 2 UAUCCAUUCAGCUAACGUU AaCGUuAgCuGaAUggAUAUsU 1 134

R-00S382574-000B 657 2 UAUCCAUUCAGCUAACGUU B UAUCCAUUCAGCUAACGUUUSU B 1129

R-008382574-000B 657 2 UAUCCAUUCAGCUAACGUU AaCGUuaGCUgAAuGgAUAUsU 1 135

R-008382217-000U 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1 136

R-008382217-OOOU 657 2 UAUCCAUUCAGCUAACGUU AACGuuAGcuGAAuGGAuAUsU 1 1 13

R-008382195-OOOF 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1 136

R-O08382195-0O0F 657 2 UAUCCAUUCAGCUAACGUU AACGuuAGcuGAAuGGAuAUsU 1 115

R-008382285-0OOY 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1 136

R-008382285-000Y 657 2 UAUCCAUUCAGCUAACGUU AACgUUagCUgaaUggaUaUsU 11 16

R-008382290-000X 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1 136

R-008382290-000X 657 2 UAUCCAUUCAGCUAACGUU AACGUUAGCUGAAUGGAUAUsU 1 117

R-0O838222O-O00A 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1136

R-008382220-OOOA 657 2 UAUCCAUUCAGCUAACGUU AACGUUAGCUGAAUGGAUAUsU 1 1 18

R-008382184-000E 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1 136

R-008382184-000E 657 2 UAUCCAUUCAGCUAACGUU AACgUUagCUgaaUggaUaUsU 1 1 19

R-008382521-000N 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1136

R-008382521-000N 657 2 UAUCCAUUCAGCUAACGUU AACgUUAGcugaAuGGaUAUsU 1137

R-008382535-OOOR 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1136

R-008382535-OOOR 657 2 UAUCCAUUCAGCUAACGUU AACgUUAGCUGAaugGAuaUsU 1138

R-008382537-OOOH 657 2 UAUCCAUUCAGCUAACGUU aaCGUUagCUGAAuGgAuAUsU 1 139

R-008382537-000H 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1 136

R-008382538-000S 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1 136

R-008382538-000S 657 2 UAUCCAUUCAGCUAACGUU AaCgUUAGCUGaauGGAuAUsU 1 140

R-008382563-000A 657 2 UAUCCAUUCAGCUAACGUU aacgUuaGCUGAAugGaUAUsU 1 141

R-008382563-O OA 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1 136

R-008382572-OOOJ 657 2 UAUCCAUUCAGCUAACGUU B uAuccAuucAGcuAAcGuuUsU B 1 136

R-008382572-000J 657 2 UAUCCAUUCAGCUAACGUU AACGUUAgcUGAAuGgaUaUsU 1142

R-008382584-000U 657 2 UAUCCAUUCAGCUAACGUU B UaUCca WCaGj:u^4CgUuUsU B 1 143

R-008382584-0OOU 657 2 UAUCCAUUCAGCUAACGUU AACGuuAGcuGAAuGGAuAUsU 1113

R-008382636-000J 657 2 UAUCCAUUCAGCUAACGUU B UaUCC/iUi/cAGC 4aCGuUUsU B 1144

R-0O8382636-0O0J 657 2 UAUCCAUUCAGCUAACGUU AACGuuAGcuGAAuGGAuAUsU 1 113

R-008382644-000 J 657 2 UAUCCAUUCAGCUAACGUU B U^ t/CCAUUC/lGCt/AAcgi7uUsU B 1 145

R-008382644-000J 657 2 UAUCCAUUCAGCUAACGUU AACGuuAGcuGAAuGGAuAUsU 1 113

R-008382422-OOOM 657 2 UAUCCAUUCAGCUAACGUU AacGUUaGcUGAaUGGAUAUsU 1 125

R-008382449-OOOH 657 2 UAUCCAUUCAGCUAACGUU B UAUcCaU CagCUAaCgUUUs B 1 147

R-008382449-000H 657 2 UAUCCAUUCAGCUAACGUU AACGUUAscUGAAuGeaUaUsU 1 142

R-008382455-000R 657 2 UAUCCAUUCAGCUAACGUU B Ua/yCC UUCaGCf/AacguUUsU B 1 154

R-0083824S5-000R 657 2 U AUCCAU UCAGCUA ACGUU AaCGUuAgCuGaAUggAUAUsU 1134

R-008382470-000G 657 2 UAUCCAUUCAGCUAACGUU B UaUCC4uuCagCU 4CGUUUsU B 1 156

R-008382470-OOOG 657 2 UAUCCAUUCAGCUAACGUU AACGuUAGcugAaUggAUAUsU 1 133

R-008382476-000J 657 2 UAUCCAU UCAGCUAACGUU aacgUuaGCUGAAugGaUAUsU 1 141

R-008382476-000J 657 2 UAUCCAUUCAGCUAACGUU B i/aUcCAut/C4gcUaaCguUUsU B 1 158

R-008382479-O0OK 657 2 UAUCCAUUCAGCUAACGUU aaCGuuAGCugAAUgGaUAUsU 1 132

R-008382479-O0OK 657 2 UAUCCAUUCAGCUAACGUU B L uCCAUUCAGCUa ICGut/UsU B 1 160

R-00838249I-000A 657 2 UAUCCAUUCAGCUAACGUU aaCguUAGCUgAAuGgAuaUsU 1120

R-008382491-OOOA 657 2 UAUCCAUUCAGCUAACGUU B C/aUCCalVUc^gCuaacgUUUsU B 1 150

R-008382494-000B 657 2 UAUCCAUUCAGCUAACGUU aACGUuAgCUgAauGGaUAUsU 1 121

R-008382494-000B 657 2 UAUCCAUUCAGCUAACGUU B VA UCc&VUCA gCUa^CeWUsU B 1153

R-008382241-OOOU 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 161

R-008382241-OOOU 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 162

R-O08382269-O00Y 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 163

R-008382269-000 Y 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 162

R-008382256-OOOE 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 163

R-008382256-OOOE 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 164

R-008382274-000X 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 163

R-008382274-OOOX 659 3 UCCAUUCAGCUAACGUUCU AGAaCgUUagCUgaaUggaUsU 1165

R-008382281-OOON 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 163

R-O08382281-OO0N 659 3 UCCAUUCAGCUAACGUUCU AGAACGUUAGCUGAAUGGAUSU 1 166

R-008382284-OOOP 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 163

R-008382284-OOOP 659 3 UCCAUUCAGCUAACGUUCU AGAACGUUAGCUGAAUGGAUSU 1 167

R-008382253-OOOD 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1163

R-008382253-000D 659 3 UCCAUUCAGCUAACGUUCU AGAaCgUUagCUgaaUggaUsU 1168

R-008382511-000W 659 3 UCCAUUCAGCUAACGUUCU agaACGUUaGcugAaUGgAUsU 1 169

R-00838251 1-000W 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 163

R-008382512-OOOE 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1163

R-008382512-000E 659 3 UCCAUUCAGCUAACGUUCU AgAaCGUuAGcUGaAUgGAUsU 1170

R-008382524-OOOP 659 ~!

-> UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 163

R-008382524-OOOP 659 3 UCCAUUCAGCUAACGUUCU AGAACGUuAgCUgaAUGGaUsU 1171

R-008382525-000Y 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 163

R-008382525-OOOY 659 3 UCCAUUCAGCUAACGUUCU AGAacgUUAGCugaAUGgAUsU 1 172

R-008382541-OOOY 659 3 UCCAUUCAGCUAACGUUCU agAACGUUAGCugAaUgGAUsU 1173

R-008382541-000Y 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 163

R-008382568-000U 659 3 UCCAUUCAGCUAACGUUCU aGAACGuUAgCUGaAugGaUsU 1 174

R-008382568-000U 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1163

R-008382223-000B 659 3 UCCAUUCAGCUAACGUUCU B uca4uuc/iGcu 4cGuucuUsU B 1 175

R-008382223-000B 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 164

R-008382209-000U 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 176

R-008382209-000U 659 3 UCCAUUCAGCUAACGUUCU AGAACGUUAGCUGAAUGGAUSU 1177

R-008382206-000T 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 11.78

R-008382206-O0OT 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 162

R-008382212- OOA 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1178

R-008382212-000A 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1164

R-008382160-OOOK 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUsU B 1 178

R-008382160-OOOK 659 3 UCCAUUCAGCUAACGUUCU AGAaCgUUagCUgaaUggaUsU 1 165 .-008382246-OOOM 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUSU B 1 178

R-008382246-000M 659 3 UCCAUUCAGCUAACGUUCU AGAACGUUAGCUGAAUGGAUsU 1 166

R-008382214-000T 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUSU B 1 178

R-008382214-000T 659 3 UCCAUUCAGCUAACGUUCU AGAACGUUAGCUGAAUGGAUsU 1 167

R-008382267-000F 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUSU B 1 178

R-008382267-OOOF 659 3 UCCAUUCAGCUAACGUUCU AGAaCgUUagCUgaaUggaUsU 1 168

R-008382508-OOOP 659 3 UCCAUUCAGCUAACGUUCU agaACgUuaGCU¾aAuGGAUsU 1 179

R-008382508-OOOP 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUSU B 1 178

R-008382509-000 Y 659 3 UCCAUUCAGCUAACGUUCU aGaACGUUagcUGaAUGGAUsU 1 180

R-0O83825O9-O00Y 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUSU B 1 178

R-008382534-OOOG 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUSU B 1 178

R-008382534-000G 659 3 UCCAUUCAGCUAACGUUCU AgaACgUuAgcUGaaUGGAUsU 1 181

R-008382557-000T 659 3 UCCAUUCAGCUAACGUUCU agAAcijuUaGCUcaAUGaAUsU 1 182

R-O08382557-0OOT 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUSU B 1 178

R-008382560-000Z 659 3 UCCAUUCAGCUAACGUUCU agaacGUUAgCuGaAUGGaUsU 1 183

R-008382560-OOOZ 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUSU B 1 178

R-008382569-000C 659 3 UCCAUUCAGCUAACGUUCU B UCCAUUCAGCUAACGUUCUUSU B 1 178

R-008382569-000C 659 3 UCCAUUCAGCUAACGUUCU AGAAcGUUaGCuGaaUggAUsU 1 184

R-O08382215-00OB 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 185

R-008382215-000B 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 162

R-008382204-0OOA 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 185

R-008382204-OOOA 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 164

R-008382216-OOOK 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1185

R-008382216-000K 659 3 UCCAUUCAGCUAACGUUCU AGAaCgUUagCUgaaUggaUsU 1 165

R-008382260-000V 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 185

R-008382260-000V 659 3 UCCAUUCAGCUAACGUUCU AGAACGUUAGCUGAAUGGAUsU 1166

R-00838226 l-OOOD 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 185

R-O08382261-000D 659 3 UCCAUUCAGCUAACGUUCU AGAACGUUAGCUGAAUGGAUsU 1 167

R-008382199-000R 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 185

R-008382199-000R 659 3 UCCAUUCAGCUAACGUUCU AGAaCgUUagCUgaaUggaUsU 1 168

R-008382510-OO M 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1185

R-O0838251O-O0OM 659 3 UCCAUUCAGCUAACGUUCU AsaACeUUAGCUgAAuGgAUsU 1 186

R-O08382518-O00G 659 3 UCCAUUCAGCUAACGUUCU aGAACguUAgCuGAAUgGaUsU 1 187

R-008382518-000G 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 185

R-O08382526-O0OG 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1185

R-008382526-000G 659 3 UCCAUUCAGCUAACGUUCU AGaACGUuaGcUGaaugGaUsU 1 188

R-008382533-000Y 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 185

R-0O8382533-O0OY 659 3 UCCAUUCAGCUAACGUUCU AGaaCGuUAGCueaauGGAUsU 1189

R-008382558-000B 659 3 UCCAUUCAGCUAACGUUCU agaacgUUAGCUGAAUGGaUsU 1 190

R-OO8382558-O0OB 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 185

R-008382559-00OK 659 3 UCCAUUCAGCUAACGUUCU B uccAuucAGcuAAcGuucuUsU B 1 185

R-008382559-000K 659 3 UCCAUUCAGCUAACGUUCU AgAaCgUuAGCUGaAUGgAUsU 1 191

R-008382603-000F 659 3 UCCAUUCAGCUAACGUUCU B UCCaVUCAgC AacGUyCUUsU B 1 192

R-008382603-000F 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 162

R-008382616-000Z 659 3 UCCAUUCAGCUAACGUUCU B uOCaUUC^GCC a^CGXJUCuUsU B 1193

R-008382616-000Z 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 162

R-008382634-000S 659 3 UCCAUUCAGCUAACGUUCU B (/CCauC CaeCi.¾acei/(/CUUsU B 1 194

R-008382634-000S 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 162

R-008382581-OOOT 659 3 UCCAUUCAGCUAACGUUCU B UcCaUuCagCUaaCGWcUUsU B 1 195

R-00838258 l-O OT 659 3 UCCAUUCAGCUAACGUUCU AGAAcGuuAGcuGAAuGGAUsU 1 164

R-008382601-000N 659 3 UCCAUUCAGCUAACGUUCU B UCC^UUCagCua^cgUUCuUsU B 1 196

R-O08382404-000V 659 3 UCCAUUCAGCUAACGUUCU B UCCd JUCAgCVAzcGlJOOWsO B 1 192

R-008382404-OOOV 659 3 UCCAUUCAGCUAACGUUCU AgAaCgUuAGCUGaAUGgAUsU 1 191

R-008382431-000W 659 3 UCCAUUCAGCUAACGUUCU B uCc/iul aGCUaaCGi/UCUUsU B 1200

R-008382431-OOOW 659 3 UCCAUUCAGCUAACGUUCU AGaaCGuUAGCugaauGGAUsU 1 189

R-008382434-OOOX 659 3 UCCAUUCAGCUAACGUUCU aGAACeuUAgCuGAAUgGaUsU 1 187

R-008382434-000X 659 3 UCCAUUCAGCUAACGUUCU B UcCaUuCagCUaaCGWcUUsU B 1 195

R-008382443-000F 659 3 UCCAUUCAGCUAACGUUCU B uCCaUt/C4GCi/a^CGXJUCuUsU B 1193

R-008382443-000F 659 3 UCCAUUCAGCUAACGUUCU AgAaCGUuAGcUGaAUeGAUsU 1 170

R-O08382485-00OT 659 3 UCCAUUCAGCUAACGUUCU B VCCAUUcAgCU&ACgmC JsU B 1 199

R-008382485-000T 659 3 UCCAUUCAGCUAACGUUCU AGAACGUuAgCUgaAUGGaUsU 1 171

R-008382488-000U 659 3 UCCAUUCAGCUAACGUUCU B i/CCaiAJC^GCi/A^CGWCUUsU B 1208

R-008382488-000U 659 3 UCCAUUCAGCUAACGUUCU AGAAcGUUaGCuGaaUggAUsU 1 184 wherein:

A, C, G, and U = ribose A, C, G or U

a, g, c and u = 2^,-deoxy-2^!-fluoro A, G, C or U

A, U, C and G = 2'-0-methyl (2'-OMe) A, U, C, or G

A, U, C, and G = deoxy A, U, C, or G

B = inverted abasic

T = thymidine

s = phosphorothioate linkage

Further Synthesis Steps for Commercial Preparation

[0328J Once analysis indicates that the target product purity has been achieved after the annealing step, the material is transferred to the tangential flow filtration (TFF) system for concentration and desalting, as opposed to doing this prior to the annealing step.

[0329] Ultrafiltration: The annealed product solution is concentrated using a TFF system containing an appropriate molecular weight cut-off membrane. Following concentration, the product solution is desalted via diafiltration using Milli-Q water until the conductivity of the filtrate is that of water.

[0330] Lyophilization: The concentrated solution is transferred to a bottle, flash frozen and attached to a lyophilizer. The product is then freeze-dried to a powder. The bottle is removed from the lyophilizer and is now ready for use. Initial Screening Protocol (96-Well Plate Transfections)

Cell Culture Preparation:

[0331] Human hepatoma cell line, HepG2, and rhesus kidney epithelial cell line, LLC-MK2 Derivative, were grown in modified Eagle's medium supplemented with 10% fetal bovine serum, 100μg/mL streptomycin, lOOU/mL penicillin, and 1 % sodium bicarbonate.

Transfection and Screening

[0332] Cells were plated in all wells of tissue-culture treated, 96-welI plates at a final count of 3500 cells/well in ΙΟΟμί of the culture media. For duration assays, HepG2 cells were plated at 1200 cells/well in ΙΟΟμΙ, medium. The cells were cultured for overnight after plating at 37°C in the presence of 5% C0₂.

[0333] On the next day. complexes containing siNA and RNAiMax (Invitrogen) were created as follows. A solution of RNAiMax diluted 33-fold in OPTI-MEM was prepared. In parallel, solutions of the siNAs for testing were prepared to a final concentration of 120 nM in OPTI-MEM. After incubation of RNAiMax/ΟΡΤΐ-ΜΕΜ solution at room temperature for 5 min, an equal volume of the siNA solution and the RNAiMax solution were added together for each of the siNAs.

[0334] Mixing resulted in a solution of siNA/RNAiMax where the concentration of siNA was 60 nM. This solution was incubated at room temperature for 20 minutes. After incubation, 20 μΐ, of the solution was added to each of the relevant wells. The final concentration of siNA in each well was 10 nM and the final volume of RNAiMax in each well was 0.3ul.

[0335] For low concentration screen, siNAs were transfected at 500 pM per well. For 12- point dose response curve studies, the siNA series are 6-fold serial dilution starting at 30nM or 4- fold serial dilution starting at 40nM. All transfections were set up as multiple biological replicates.

[0336] The time of incubation with the RNAiMax-siNA complexes was 24 hours and there was no change in media between transfection and harvesting for screening and dose response curve studies. For duration assays, the time of incubation with the RNAiMax-siNA complexes was 24 and 120 hours. There was no change in media between transfection and harvesting for 24-hour time point. Media was replaced with fresh media 72 hours after transfection for 120- hour time point.

Cells-to-Ct and reverse transcription reactions

[0337] The culture medium was aspirated and discarded from the wells of the culture plates at the desired time points. The transfected cells were washed once with 50uL DPBS solution per well. Fifty microliters per well of the Lysis Solution from the TaqMan® Gene Expression Cells- to-CT™ Kit (Applied Biosystems, Cat# 4399002) supplemented with DNase I was added directly to the plates to lyse the cells. Five microliters per well of Stop Solution from the same kit was added to the plates to inactivate DNase 1 5 min. later. The lysis plates were incubated for at least 2 min. at room temperature. The plates can be stored for 2 hours at 4°C, or -80 °C for two months.

[0338] Each well of the reverse transcription plate required lOuL of 2X reverse transcriptase buffer, luL of 20X reverse transcription enzyme and 2uL of nuclease-free water. The reverse transcription master mix was prepared by mixing 2X reverse transcription buffer, 20X reverse transcription enzyme mix, and nuclease-free water. 13uL of the reverse transcription master mix was dispensed into each well of the reverse transcription plate (semi-skirted). A separate reverse transcription plate was prepared for each cell plate. A separate reverse transcription plate was prepared for each cell plate. Seven microliters per lysate from the cell lysis procedure described above was added into each well of the reverse transcription plate. The plate was sealed and spun on a centrifuge (lOOOrpm for 30 seconds) to settle the contents to the bottom of the reverse transcription plate. The plate was placed in a thermocycler at 37 °C for 60 min, 95 °C for 5 min, and 4 °C until the plate is removed from the thermocycler. Upon removal, if not used immediately, the plate was frozen at -20 °C.

[0339] For duration assays, a similar protocol was followed, however, the cells were lysed 1 and 5 days after transfection. cDNA was generated using Cells-to-Ct™ Kit (Applied Biosystems). Quantitative RT-PCR (Taqman)

[0340] A series of probes and primers were used to detect the various mRNA transcripts of the genes of MAP l and GAPDH. All Taqman probes and primers for the experiments here-in described were supplied as pre-validated sets by Applied Biosystems, Inc. (see Table 2).

[0341]

Table 2: Probes and primers used to carry out Real-Time RT/PCR (Taqman) reactions for

[0342] The assays were performed on an ABI 7900 instrument, according to the manufacturer's instructions. A TaqMan Gene Expression Master Mix (provided in the Cells-to- CT™ Kit, Applied Biosystems, Cat # 4399002) was used. The PCR reactions were can-ied out at 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles at 95 °C for 15 sees and 60 °C for 1 min.

[0343] Within each experiment, the baseline was set in the exponential phase of the amplification curve, and based on the intersection point of the baselines with the amplification curve, a Ct value was assigned by the instrument.

Calculations

[0344] The expression level of the gene of interest and % inhibition of gene expression (%KD) was calculated using Comparative Ct method:

ACt = Ct Target ~ Ct GAPDH

AACt (log2(fold change)) - ACt (Target siNA) ~ ACt (NTC)

Relative expression level % KD = 100 x (l - 2™)

[0345] The non-targeting control siNA was, unless otherwise indicated, chosen as the value against which to calculate the percent inhibition (knockdown) of gene expression, because it is the most relevant control.

[0346] Additionally, only normalized data, which reflects the general health of the cell and quality of the RNA extraction, was examined. This was done by looking at the level of two different mR As in the treated cells, the first being the target mRNA and the second being the normalizer mRNA. This allowed for elimination of siNAs that might be potentially toxic to cells rather than solely knocking down the gene of interest. This was done by comparing the Ct for GAPDH in each well relative to the GAPDH Ct for the entire plate.

[0347] All calculations of IC50S were performed using R.2.9.2 software. The data were analyzed using the sigmoidal dose-response (variable slope) equation for simple ligand binding. In all of the calculations of the percent inhibition (knock-down), the calculation was made relative to the normalized level of expression of the gene of interest in the samples treated with the non-targeting control (Ctrl siNA) unless otherwise indicated.

Results:

[0348] The MAPK1 siNAs were designed and synthesized as described previously. Various siNAs were screened in HepG2 cells at 2 transfection concentrations, l OnM and 500pM. The log 2(fold change) in MAPK1 gene expression data upon treatment with various modified MAPKl siNAs in human cells is shown in Table 3a and b. Each screen was performed at 24 hrs. Quantitative RT-PCR was used to assess the level of MAPKl mRNA and the data were normalized to the expression level of GAPDH (an ubiquitously expressed 'house-keeping' gene). Each treatment was then normalized against the non-MAPKl targeting control.

Table 3a: Primary screening data in HepG2 Cells (n = 2-4) q5 l OnMs, recorded as log 2(fold change) in MAPKl gene expression.

R-008352126-000A 3.57 0.26

R-008351982-000D 3.50 0.02

R-008352144-000T 3.47 0.09

R-008352000-000D 3.45 0.21

R-008351922-OOOZ 3.37 0.15

R-008352123-000Z 3.36 0.24

R-008352165-000L 3.35 0.21

R-008352063-000J 3.32 0.31

R-008352153-000B 3.32 0.05

R-008351928-000B 3.30 0.22

R-008352105-000G 3.29 0.06

R-008352099-000N 3.29 0.01

R-008274589-O00J 3.25 0.29

R-008352117-000S 3.25 0.33

R-008351919-000T 3.24 0.09

R-008351934-000J 3.24 0.01

R-008352006-000F 3.23 0.18

R-008351970-000U 3.12 0.05

R-008351967-000M 3.12 0.00

R-008274691-000S 3.1 1 0.24

R-O08352O90-O0O 3.1 1 0.18

R-008352147-000U 3.09 0.09

R-008352039-000J 3.08 0.15

R-008351931-000H 3.08 0.04

R-008352045-000S 3.08 0.19

R-008293708-000P 3.08 0.18

R-008293597-000T 3.06 2.97

R-008352132-000H 3.05 0.03

R-008293642-000X 3.03 0.05

R-008352075-000U 3.01 0.18

R-008352093-000L 3.01 0.05

R-008352168-000M 3.00 0.05

R-008293798-000W 2.99 0.13

R-008352120-000Y 2.98 0.1 1

R-008274682-000H 2.98 0.07

R-008352003-000E 2.97 0.06

R-008352051-O00Z 2.97 0.18

R-008352087-000D 2.95 0.01

R-008351940-000S 2.94 0.11

R-008274657-OOOZ 2.93 0.49

R-008352012-000N 2.93 0.05

R-008274992-000E 2.93 0.01

R-008352150-000A 2.93 0.06

R-008274734-000Y 2.92 0.00

R-008275033-000R 2.92 0.05 R-008352129-000B 2.91 0.28

R-008352015-000P 2.91 0.21

R-008275097-000E 2.91 0.08

R-008352069-000L 2.90 0.07

R-008352108-000H 2.89 0.10

R-008293612-000V 2.89 0.06

R-008274982-000M 2.87 0.18

R-008351925-000A 2.85 0.20

R-008293834-000S 2.85 0.13

R-008351988-000F 2.85 0.12

R-008274597-000J 2.84 0.06

R-008351976-000W 2.83 0.10

R-008351943-000T 2.83 0.32

R-008352135-000J 2.82 0.01

R-008352102-000F 2.82 0.04

R-008352024-000Y 2.82 0.15

R-008352042-O00R 2.81 0.23

R-008293639-000R 2.81 0.07

R-008352018-OOOR 2.81 0.04

R-008293822-000G 2.80 0.08

R-008293774-000B 2.80 0.15

R-008293726-000G 2.80 0.08

R-008274640-000W 2.79 0.02

R-008351961-00OK 2.78 0.18

R-008275133-000A 2.77 0.48

R-008293603-000L 2.76 0.05

R-008293840-0OOZ 2.75 0.10

R-008351952-000B 2.75 0.07

R-008293630-000M 2.73 0.12

R-008293756-000J 2.72 0.24

R-008352021-000X 2.72 0.12

R-008293711-000W 2.71 0.06

R-008293843-000A 2.71 0.05

R-008274632-000W 2.70 0.1 1

R-008352048-000T 2.69 0.42

R-008352096-OOOM 2.68 0.08

R-0O8352138-O0OK 2.68 0.17

R-008293675-000A 2.66 0.19

R-008275052-OOOS 2.66 0.18

R-008352141-O0OS 2.66 0.04

R-008293768-OOOU 2.65 0.21

R-008274624-000W 2.65 0.04

R-008293651 -000F 2.64 0.16

R-00835211 1-000P 2.63 0.04

R-008293687-000K 2.63 0.01 R-008352033-000G 2.61 0.17

R-008351955-000C 2.58 0.16

R-008352066-OQO 2.58 0.03

R-008351985-000E 2.57 0.29

R-008293666-000S 2.56 0.05

R-008352054-000A 2.56 0.09

R-008352009-OOOG 2.56 0.17

R-008351946-000U 2.55 0.35

R-008293759-000K 2.55 0.41

R-008352081-000B 2.55 0.22

R-008293765-000T 2.54 0.48

R-008293762-000S 2.54 0.21

R-O08274666-000H 2.50 0.19

R-O08293795-00OV 2.50 0.09

R-008293615-000W 2.49 0.09

R-008293660-000P 2.48 0.22

R-008293699-000V 2.47 0.80

R-008293738-000S 2.47 0.09

R-008352072-000T 2.47 0.25

R-008293777-000C 2.46 0.19

R-008293663-000R 2.46 0.07

R-008293627-OOOF 2.46 0.06

R-O08352030-O0OF 2.46 0.12

R-0O8293678-O0OB 2.43 0.03

R-008293621-000D 2.43 0.07

R-008293693-000T 2.42 0.10

R-008352027-000Z 2.41 0.08

R-O08293783-000K 2.41 0.13

R-008293645-000Y 2.41 0.16

R-008351979-000X 2.40 0.14

R-008352060-000H 2.40 0.26

R-008293837-000T 2.39 0.25

R-008293825-000H 2.38 0.46

R-008293780-000J 2.38 0.13

R-008351937-000 2.33 0.25

R-008274799-000W 2.33 0.05

R-008293744-000Z 2.31 0.25

R-008275042-OOOZ 2.30 0.38

R-008293810-OOOX 2.29 0.10

R-O08275061-00OA 2.26 0.02

R-008293624-OOOE 2.26 0.02

R-0O8293729-000H 2.25 0.27

R-008293681-000H 2.21 0.06

R-008293813-000Y 2.19 0.01

R-008274752-000R 2.17 0.13 R-008352156-000C 2.14 0.09

R-008352084-000C 2.12 0.08

R-008274717-000P 2.10 0.12

R-008274709-000P 2.07 0.26

R-008293714-000X 2.06 0.14

R-008352162-000K 2.06 0.12

R-00829369O-OO0S 2.02 0.54

R-008274648-000R 2.01 0.22

R-O08274725-00OP 2.01 0.14

R-008293732-000P 2.01 0.12

R-008293753-000H 1.98 0.14

R-008293696-000U 1.93 0.04

R-008351958-000D 1.87 0.15

R-008351949-000V 1.86 0.18

R-008293633-000N 1.83 0.18

R-008274742-000Y 1.80 0.09

R-008293831-000R 1.74 0.00

R-008352159-000D 1.72 0.18

R-008293792-000U 1.65 0.02

R-008293669-000T 1.64 0.07

R-008293816-000Z 1.64 0.1 1

R-008275010-000E 1.60 0.00

R-008293720-000E 1.49 1.01

R-008274605-00OV 1.49 0.10

R-008274789-OOOD 1.46 0.12

R-008275020-OOOX 1.38 0.01

R-008293717-000Y 1.36 0.41

R-008351991 -000M 1.36 0.06

R-008352057-000B 1.34 0.13

R-008275123-000H 1.32 0.19

R-008293684-000J 1.30 0.1 1

R-008293636-000P 1.25 0.05

R-008293807-000R 1.23 0.09

R-008293702-000M 1.21 0.02

R-008274779-000L 1.18 0.08

R-008293618-000X 1.15 0.10

R-008275113-OOOR 1.14 0.21

R-008351973-OOOV 1.09 0.09

R-008293594-000S 1.09 0.33

R-008293741 -000Y 0.93 0.05

R-008275105-000R 0.92 0.04

R-008293606-000M 0.75 0.06

R-008293789-000M 0.75 0.03

R-008274761-000Z 0.75 0.06

R-008275087-000M 0.73 0.00 R-008274701-000V 0.73 0.00

R-008274581-000P 0.66 0.10

R-008274615-000M 0.66 0.04

R-008293801 -O00N 0.65 0.02

R-008352O36-O00H 0.64 0.10

R-008293723-OOOF 0.58 0.1 1

R-008293804-000P 0.54 0.47

R-008274770-O00H 0.53 0.19

R-008293600-000K 0.53 0.35

R-008275078-O0OD 0.45 0.16

R-008274675-000S 0.39 0.04

R-O08293657-O00H 0.39 0.02

R-008293747-000A 0.37 0.06

R-008275070-000J 0.31 0.21

R-008293735-000R 0.28 0.02

R-008293771-000A 0.25 0.03

R-008293750-000G 0.23 0.45

R-008293819-000A 0.20 0.42

R-008352078-OOOV 0.18 0.1 1

R-00S275001 -000W 0.16 0.04

R-008293648-000Z 0.07 0.13

R-008293654-000G 0.04 0.05

R-O08293609-O00N -0.03 0.10

R-008351997-000P -0.03 0.18

R-0O8293828-000J -0.04 0.06

R-008293672-000Z -0.10 0.35

R-008293786-000L -0.22 0.09

R-008293705-O00N -1.21 0.01

Table 3b: Primary screening data in HepG2 Cells (n = 2-4) at 500pM, recorded as log 2(fold change) in MAP l gene expression.

R-008352021-OOOX 2.13 0.83

R-008352123-0O0Z 2.13 0.33

R-0083521 14-000R 2.06 0.38

R-008352120-000Y 2.05 0.35

R-008351982-000D 2.00 0.42

R-008352105-000G 1.96 0.22

R-008351955-OOOC 1.94 0.51

R-008352135-000J 1.94 0.17

R-0083521 1 1-000P 1.92 0.36

R-008352102-OOOF 1.90 0.59

R-008352000-000D 1.83 0.22

R-008352075-000U 1.81 0.39

R-008352081-000B 1.81 0.30

R-008352042-000R 1.80 0.22

R-008352090-000K 1.78 0.27

R-008351964-000L 1.77 0.36

R-008352087-000D 1.76 0.22

R-008351967-000M 1.76 0.33

R-008352147-000U 1.74 0.16

R-008352006-000F 1.67 0.37

R-008352003-OOOE 1.64 0.31

R-008352093-0O0L 1.62 0.33

R-008351970-000U 1.58 0.41

R-008352024-OOOY 1.52 0.27

R-008352018-000R 1.51 0.22

R-008352033-000G 1.42 0.32

R-008352048-OOOT 1.37 0.45

R-008352132-000H 1.28 0.24

R-008351919-000T 1.27 0.37

R-008351940-000S 1.23 0.17

R-008352030-000F 1.21 0.21

R-008352009-000G 1.17 0.36

R-008352129-000B 1.02 0.53

R-008352039-000J 0.94 0.42

R-008352027-000Z 0.93 0.32

R-008351952-000B 0.88 0.20

R-008352051-000Z 0.86 0.10

R-008352099-000N 0.83 0.35

R-008352144-000T 0.81 0.17

R-008352096-000M 0.80 0.17

R-008351937-OOOK 0.76 0.44

R-008352153-000B 0.76 0.12

R-0083521 17-OO0S 0.71 0.12

R-008352168-OOOM 0.71 0.30

R-008352138-000K 0.70 0.27 R-008352150-000A 0.69 0.27

R-008352045-000S 0.65 0.15

R-008352084-O00C 0.64 0.26

R-008351961 -000K 0.61 0.44

R-008352066-000K 0.60 0.42

R-008351946-000U 0.60 0.18

R-008352162-O00K 0.58 0.23

R-008351931-000H 0.58 0.19

R-008351925-000A 0.57 0.29

R-008351979-000X 0.55 0.06

R-008352072-000T 0.54 0.31

R-008352060-000H 0.50 0.43

R-008352069-OOOL 0.49 0.17

R-008352054-OO0A 0.48 0.19

R-008352015-000P 0.47 0.22

R-008352012-000N 0.45 0.14

R-008351985-000E 0.41 0.16

R-008351988-000F 0.40 0.47

R-008352156-000C 0.34 0.18

R-008351949-000V 0.32 0.26

R-008351922-000Z 0.31 0.14

R-008352057-000B 0.23 0.14

R-008352141-000S 0.16 0.10

R-O08351958-00OD 0.11 0.06

R-008351991 -OO0M 0.10 0.13

R-008352036-000H 0.06 0.1 1

R-008351997-000P 0.06 0.06

R-008352078-000V 0.04 0.14

R-008351973-000V 0.01 0.07

R-008352159-000D 0.00 0.23

R-008352108-000H -0.14 0.27

[0349] Select sequences in Tables 3a and 3b were retested for knockdown represented as .log 2(fold change). The results are shown in Table 3c below.

Table 3c: Primary screening data in HepG2 Ceils (n = 2-4) at ΙΟηΜ and 500pM, recorded as log 2(fold change) in MAPK1 gene expression.

R-008293783-000K 3.67 0.22 3.62 1.12

R-008293603-000L 3.04 0.32 3.59 0.42

R-008274682-000H 4.40 0.17 3.55 0.03

R-008293813-000Y 3.81 0.09 3.42 0.70

R-008293642-000X 3.56 0.04 3.39 0.53

R-008274691-OO0S 4.1 1 0.24 3.34 0.46

R-008293822-000G 3.48 0.08 3.32 0.58

R-008293834-OO0S 3.26 0.08 3.30 0.24

R-008293663-000R 3.19 0.12 3.10 0.59

R-008274734-000Y 3.03 0.12 3.09 0.76

R-008293765-000T 3.67 0.10 3.06 0.08

R-008293708-000P 3.04 0.08 3.04 0.06

R-008293615-000W 3.15 0.37 2.99 0.18

R-008275097-000E 2.64 0.31 2.98 0.34

R-008293798-000W 3.70 0.08 2.97 0.29

R-008293699-000V 3.37 0.04 2.96 0.70

R-008293729-000H 3.19 0.10 2.94 0.07

R-008293777-O0OC 3.61 0.14 2.92 0.51

R-008293756-OO0J 3.52 0.01 2.90 0.09

R-008293738-OOOS 3.17 0.02 2.74 0.20

R-008293639-000R 3.09 0.21 2.66 0.46

R-008293843-000A 2.61 0.18 2.64 0.01

R-008274640-000W 3.43 0.17 2.63 0.06

R-008293810-OOOX 2.67 0.49 2.62 0.95

R-008293666-000S 3.10 0.23 2.61 0.27

R-008293762-000S 2.90 0.41 2.60 0.70

R-008275052-000S 2.95 0.13 2.57 0.54

R-008293693-000T 3.03 0.01 2.56 0.46

R-008275133-OOOA 2.64 0.25 2.55 0.57

R-008293675-OOOA 3.08 0.04 2.53 0.51

R-008293687-000K 2.69 0.29 2.49 0.09

R-008293768-000U 3.01 0.25 2.48 0.29

R-008293690-000S 2.67 0.19 2.46 0.13

R-008293627-000F 3.16 0.54 2.46 0.75

R-008293660-000P 3.32 0.02 2.41 0.44

R-008274752-0O0R 3.77 1.14 2.40 0.09

R-008293732-OO0P 2.46 0.12 2.34 1.22

R-008293681-O0OH 2.69 0.30 2.30 0.18

R-008293744-000Z 3.06 0.01 2.29 0.30

R-008293837-000T 3.06 0.06 2.29 0.18

R-008293774-000B 2.96 0.10 2.26 0.32

R-008293792-000U 3.18 0.06 2.22 0.83

R-008274717-000P 2.57 0.21 2.13 0.69

R-008293651-000F 2.35 0.14 2.12 0.46

R-008293678-000B 2.39 0.24 2.10 0.96 R-008274648-000R 0.72 2.97 2.04 0.26

R-008293840-000Z 2.91 0.06 2.02 0.57

R-008275123-000H 1.99 0.01 1.81 0.27

R-008274597-000J 2.45 0.20 1.79 0.43

R-008293780-000J 2.62 0.1 1 1.79 0.69

R-008275010-000E 2.22 0.23 1.75 0.31

R-00829371 1 -O0OW 3.10 0.29 1.63 0.39

R-008293624-OOOE 3.46 0.01 1.58 0.31

R-008293594-00OS 2.62 0.22 1.57 0.12

R-008293714-000X 2.34 0.83 1.48 0.37

R-008274761 -000Z 2.02 0.17 1.47 0.42

R-008293702-000M 2.29 0.09 1.43 0.23

R-008274789-OOOD 2.37 0.62 1.36 0.17

R-008293831-OOOR 2.26 0.05 1.16 0.21

R-008293648-O00Z 0.45 0.30 1.15 0.33

R-008275087-000M 1.65 0.33 1.1 1 0.24

R-008293645-000Y 3.08 0.03 1.06 0.31

R-008274701-000V 1.65 0.01 0.99 0.09

R-008293807-000R 2.47 0.09 0.80 0.19

R-008274779-000L 2.38 0.09 0.72 0.22

R-008293684-OOO.T 2.49 0.03 0.59 1.26

R-008293597-000T 2.04 0.32 0.55 0.09

R-008275078-000D 1.36 0.10 0.53 0.06

R-0082751 13-000R 2.34 0.54 0.43 0.51

R-008274605-000V 1.75 0.24 0.1 1 0.53

[0350] A subset of siNAs from Table 3a and Table 3b having a large log2(fold change) in the primary screen were evaluated in an in vitro duration assay. The log2(fold change) (reported as AACt) after 1 and 5 days was determined. The results are shown in Table 4.

Table 4. Log2(fold change) data from in vitro duration assay.

R-008352021-000X 2.85 0.22 2.57 0.26

R-008293699-000V 2.23 0.38 2.55 0.28

R-008293765-000T 2.93 0.27 2.52 0.09

R-008293663-000R 2.70 0.61 2.39 0.09

R-008293768-000U 2.43 0.34 2.36 0.32

R-008293756-000J 1.73 0.40 2.35 0.83

R-008293726-000G 2.53 0.31 2.34 0.20

R-008274734-000Y 2.39 0.18 2.27 0.24

R-008293708-000P 3.19 0.42 2.25 0.24

R-008351928-000B 3.31 0.25 2.16 0.40

R-008352102-000F 3.00 0.25 2.11 0.08

R-008352024-000Y 2.59 0.18 2.06 0.13

R-008293798-000W 2.42 0.81 2.05 0.16

R-008293738-OOOS 2.36 0.38 2.03 0.19

R-008351943-000T 3.32 0.17 2.02 0.29

R-008274691-000S 3.30 0.36 1.96 0.38

R-008293660-000P 2.33 0.18 1.95 0.24

R-008352006-000F 2.78 0.34 1.95 0.19

R-008293642-000X 3.22 0.15 1.93 0.36

R-008274682-000H 2.98 0.19 1.93 0.17

R-008352063-000J 2.80 0.42 1.91 0.34

R-008293825-000H 2.55 0.22 1.88 0.29

R-008293693-000T 1.98 0.15 1.78 0.08

R-OO8293777-000C 2.57 1.11 1.69 0.15

R-008351934-000J 3.30 0.30 1.65 0.81

R-008293639-000R 2.00 0.35 1.59 0.27

R-008352123-000Z 3.28 0.43 1.57 0.48

R-008352105-000G 3.19 0.31 1.56 0.21

R-008275097-000E 2.61 0.17 1.53 0.60

R-008293729-000H 2.04 0.17 1.47 0.21

R-008293774-000B 2.31 0.25 1.46 0.23

R-008352165-000L 2.96 0.23 1.46 0.55

R-008293711-000W 2.81 0.14 1.28 0.22

R-008352075-000U 2.55 0.36 1.22 0.37

R-008352120-000Y 2.59 0.30 1.17 0.23

R-008351982-000D 3.04 0.21 1.10 0.28

[0351] Select siNAs from Table 3a and Table 3b having a large log2(fold change) were also analyzed for efficacy and potency in HepG2 and LLC-MK2 Derivative cells to ensure that these siNAs functioned well in monkey cells for primate studies. The results for these siNAs are shown in Table 5. The potency 50 is the calculated siNA transfection concentration that produces 50% target mRNA knockdown. Table 5: Dose response data for various siNAs in HepG2 -Human hepatoma and LLC-MK2 Derivative - Rhesus monkey kidney epithelial cell lines. Calculated maximum AACt is determined from the dose res onse curve.

Example 2: Determining In Vitro Serum Stability of siNAs

[0352] siNAs are reconstituted as 50μΜ to Ι ΟΟμΜ stock solution with H₂0 and added to human serum pre-warmed to 37°C to a final concentration of 2C^g/mL. The mixture is then incubated at 37°C for 0, 1 and 2 hours. At the end of each time point, the reactions are stopped by mixing with equal volume of Phenomenex Lysis-Loading Buffer. Oligonucleotides are purified in 96-well format by Phenomenex Solid Phase Extraction and lyophilized until dry with Labconco Triad Lyo-00417. The lyophilized samples are reconstituted in 150 pL of 1 mM EDTA prepared with RNase-free H₂0. The sample solutions re then diluted 5 fold with 1 mM EDTA for liquid chromatography/mass spectrometiy (LC/MS) analysis on ThermoFisher Orbitrap. Serum metabolites of the siNAs were determined based on the measured molecular weights. Example 3 : Testing of Cytokine Induction

(0353] To assess immunostimulative effects of various siNAs of the invention loaded in lipid nanoparticles (DLinDMA/Cholesterol/ S-PEG-C-DMA/DSPC in a 40/48/2/10 ratio), C57B1/6 mice are dosed with a single 3mpk dose of LNP formulated siNAs through tail vein injection. Serum or plasma samples are collected at 3 and 24 hours post-dose. The cytokine and chemokine levels in these samples is measured with the SearchLight IR Cytokine Array from Aushon Biosciences according to the manufacturer's instruction. The cytokines and chemokines measured are IL-la, IL-Ι β, IL-6, KC, IL-10, IFNy, TNF, GMCSF, ΜΙΡ-Ι β, MCP-l/JE, and RANTES.

Example 4: Efficacy Studies in Mouse.

[0354] In vivo efficacy studies were conducted in TRE-MET mice maintained at Taconic Farms. Mice were dosed IV via tail vein injections with LNP encapsulated siNAs or vehicle control using 2 different 3-week dosing schemes: one 1 mg/kg dose for 3 consecutive days or a single 6 mg kg does per week. In some experiments, the mice were co-dosed with sorafenib at a dose of 100 mg/kg QD or BID every day for 3 weeks. Total tumor burden was measured by micro-CT scan imaging. The animals were sacrificed 5 days after the last siNA dose (Day 23), and normal liver and tumor tissues from each animal were collected for RNA purification. Total RNA was purified using RNeasy 96 kit (Qiagen, Cat# 74182). cDNA was generated from total RNA using High Capacity cDNA Reverse Transcription Kit (Cat#: 4368813). Quantitative PCR reactions were performed with TaqMan Universal PCR Master Mix (Cat#: 4304437). Mouse MAPK1 TaqMan Gene Expression Assay (Mm004424795_ml ) and mouse GAPDH TaqMan Gene Expression Assay (Cat#: 4352932E) were used to monitor the mRNA level of both transcripts. The expression level of MAPK1 was normalized against GAPDH to minimize technical variations. All reverse transcription and quantitative PCR reagents are from Applied Biosystems, Inc.

[0355] As shown in Table 6 and Figure 11, treatment of TRE-MET mice with various lead siNAs of the invention resulted in tumor growth inhibition achieving tumor stasis at 3 weeks. Co-dosing with sorafenib did not result in statistically significant increased efficacy.

Table 6. Summary of in vivo studies using MAPK1 siNAs in TRE-MET mice. siNA Duplex ID CombisiNA dosing CombiLiver Tumor Tumor nation scheme nation % % phenotype reagent reagent inhibiinhibidosing tion tion

scheme

R-008274691-000S None 3xlmpk/week NA 84 33 70% reduction of

X 3 weeks tumor growth

R-OO8274691 -00OS Sorafenib 3xlmpk/week 100 mpk 85 53 tumor stasis

X 3 weeks qd (100% reduction of tumor growth)

R-008274691-000S None l 6mpk weekX NA 86 35 tumor stasis

3 weeks (100% reduction in tumor growth)

R-008274691-000S Sorafenib 1 x6mpk/week 100 mpk 84 44 97% reduction in

X 3 weeks qd tumor growth

R-008274982-000M None 1 x6mpk weekX NA 81 41 54% reduction in

3 weeks tumor growth

R-008274982-000M Sorafenib 1 x6mpk/week 100 mpk 85 49 75% reduction in

X 3 weeks bid tumor growth

Example 5: Pharmacodynamic Study in Non-Human Primates

Rhesus macaque monkeys are dosed with a single 2.5mpk dose of siNA loaded lipid nanoparticles through intravenous infusion. To monitor target mRNA knockdown, liver biopsies are performed at various time points pre- and post-dose with 16T gauge Menghini needles for about 20mg tissue per animal. Whole blood and serum/plasma is also collected at different time points pre- and post-dose to monitor potential toxicity associated with the treatments. All procedures adhere to the regulations outlined in the USDA Animal Welfare Act (9 CFR, Parts 1, 2 and 3) and the conditions specified in The Guide for Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press).

[0356] LNP formulations (DLinDMA/Cholesterol/S-PEG-C-DMA/DSPC in a 40/48/2/10 ratio) comprising the siNA are tested. Log2(fold change) in MAPKl gene expression is determined on days 3, 7, 14, and 28 days post-dosing. Pre-dose MAPKl expression levels for the monkey is measured 7 days before the first dosing.

Example 6: siNAs LNP Formulations

A. General LNP Process Description for DLinDMA Formulations: [0357] The lipid nanoparticles were prepared by an impinging jet process. The particles were formed by mixing lipids dissolved in alcohol with siNA dissolved in a citrate buffer. The mixing ratio of lipids to siNA is targeted at 45-55% lipid and 65-45% siNA. The lipid solution contained a cationic lipid, a helper lipid (cholesterol), PEG (e.g. PEG-C-DMA, PEG-DMG) lipid, and DSPC at a concentration of 5-15 mg/mL with a target of 9- 12 mg/mL in an alcohol (for example ethanol). The ratio of the lipids had a mole percent range of 25-98 for the cationic lipid with a target of 35-65, the helper lipid had a mole percent range from 0-75 with a target of 30-50, the PEG lipid has a mole percent range from 1-15 with a target of 1 -6, and the DSPC had a mole percent range of 0-15 with a target of 0- 12. The siNA solution contained one or more siNA sequences at a concentration range from 0.3 to 1 .0 mg/mL with a target of 0.3 -0.9 mg/mL in a sodium citrate buffered salt solution with pH in the range of 3.5-5. The two liquids were heated to a temperature in the range of 15-40°C, targeting 30-40°C, and then mixed in an impinging jet mixer instantly forming the LNP. The tee!D had a range from 0.25 to 1.0 mm and a total flow rate from 10-600 mL/minute The combination of flow rate and tubing ID had the effect of controlling the particle size of the LNPs between 30 and 200 nm. The solution was then mixed with a buffered solution at a higher pH with a mixing ratio in the range of 1 : 1 to 1 :3 vohvol but targeting 1 :2 vokvol. This buffered solution was at a temperature in the range of 15- 40°C, targeting 30-40°C. The mixed LNPs were held from 30 minutes to 2 hrs prior to an anion exchange filtration step. The temperature during incubating was in the range of 15-40°C, targeting 30-40°C. After incubating, the solution was filtered through a 0.8 urn filter containing an anion exchange separation step. This process used tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/minute The LNPs were concentrated and diafiltered via an ultrafiltration process where the alcohol was removed and the citrate buffer was exchanged for the final buffer solution such as phosphate buffered saline. The ultrafiltration process used a tangential flow filtration format (TFF). This process used a membrane nominal molecular weight cutoff range from 30 -500 KD. The membrane format was hollow fiber or flat sheet cassette. The TFF processes with the proper molecular weight cutoff retained the LNP in the retentate and the filtrate or permeate contained the alcohol; citrate buffer; and final buffer wastes. The TFF process is a multiple step process with an initial concentration to a siNA concentration of 1 -3 mg/mL. Following concentration, the LNPs solution was diafiltered against the final buffer for 10 -20 volumes to remove the alcohol and perform buffer exchange. The material was then concentrated an additional 1-3 fold. The final steps of the LNP process were to sterile filter the concentrated LNP solution and vial the product.

Analytical Procedure:

1) siNA concentration

[0358] The siNA duplex concentrations were determined by Strong Anion-Exchange High- Performance Liquid Chromatography (SAX-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a 2996 PDA detector. The LNPs, otherwise referred to as RNAi Delivery Vehicles (RDVs), were treated with 0.5% Triton X-100 to free total siNA and analyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4 x 250 mm) column with UV detection at 254 nm. Mobile phase was composed of A: 25 mM NaGd, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaC10₄, 10 mM Tris, 20% EtOH, pH 7.0 with a liner gradient from 0-15 min and a flow rate of 1 ml/minute. The siNA amount was determined by comparing to the siNA standard curve.

2) Encapsulation rate

[0359] Fluorescence reagent SYBR Gold was employed for RNA quantitation to monitor the encapsulation rate of RDVs. RDVs with or without Triton X-100 were used to detennine the free siNA and total siNA amount. The assay is performed using a SpectraMax M5e microplate spectrophotometer from Molecular Devices (Sunnyvale, CA). Samples were excited at 485 nm and fluorescence emission was measured at 530 nm. The siNA amount is determined by comparing to an siNA standard curve.

Encapsulation rate = (1 - free siN A/total siNA) * 100%

3) Particle size and polydispersity

[0360] RDVs containing 1 μg siNA were diluted to a final volume of 3 ml with 1 ^χ PBS. The particle size and polydispersity of the samples was measured by a dynamic light scattering method using ZetaPALS instrument (Brookhaven Instruments Corporation, Holtsville, NY). The scattered intensity was measured with He-Ne laser at 25°C with a scattering angle of 90°. 4) Zeta Potential Analysis

[0361] RDVs containing 1 μg siNA were diluted to a final volume of 2 ml with 1 raM Tris buffer (pH 7.4). Electrophoretic mobility of samples was determined using ZetaPALS instrument (Brookhaven Instruments Corporation, Holtsville, NY) with electrode and He-Ne laser as a light source. The Smoluchowski limit was assumed in the calculation of zeta potentials.

5) Lipid analysis

[0362] Individual lipid concentrations were determined by Reverse Phase High-

Performance Liquid Chromatography (RP-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a Corona charged aerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, MA). Individual lipids in RDVs were analyzed using an Agilent Zorbax SB- C I S (50 x 4.6 mm, 1.8 μιη particle size) column with CAD at 60°C. The mobile phase was composed of A: 0.1% TFA in H₂0 and B: 0.1% TFA in IPA. The gradient changed from 60% mobile phase A and 40% mobile phase B from time 0 to 40% mobile phase A and 60% mobile phase B at 1 .00 min; 40% mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobile phase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and 75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phase B from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00 min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60% mobile phase A and 40% mobile phase B at 20.00 min with a flow rate of 1 ml/minute. The individual lipid concentration was determined by comparing to the standard curve with all the lipid components in the RDVs with a quadratic curve fit. The molar percentage of each lipid was calculated based on its molecular weight.

B. General Formulation Procedure for CLinDMA Cholesterol/PEG-DMG at a ratio of 71.9:20,2:7.9.

[0363] siNA solutions were prepared by dissolving siNAs in 25mM citrate buffer (pH 4.0) at a concentration of 0.8mg/mL. Lipid solutions were prepared by dissolving a mixture of 2S- Octyl-ClinDMA, cholesterol and PEG-DMG at a ratio of 71.9:20.2:7.9 in absolute ethanol at a concentration of about lOmg/mL. Equal volume of siNA and lipid solutions were delivered with two syringe pumps at the same flow rates to a mixing T connector. The resulting milky mixture was collected in a sterile bottle. This mixture was then diluted slowly with an equal volume of citrate buffer, and filtered through a size exclusion hollow fiber cartridge to remove any free siNA in the mixture. Ultra filtration against citrate buffer (pH 4.0) was employed to remove ethanol (test stick from ALCO screen), and against PBS (pH 7.4) to exchange buffer. The final L P was obtained by concentrating to a desired volume and sterile filtered through a 0.2mm filter. The obtained LNPs were characterized in term of particle size, alcohol content, total lipid content, nucleic acid encapsulated, and total nucleic acid concentration.

C. General LNP Preparation For Various Formulations in Table 10.

[0364] siNA nanoparticle solutions in Table 10 are prepared by dissolving siNAs and/or carrier molecules in 25 mM citrate buffer (pH 4.0) at a concentration of 0.9 mg/mL. Lipid solutions are prepared by dissolving a mixture of cationic lipid (e.g., CLinDMA or DOBMA, see structures and ratios for Formulations in Table 10), DSPC, Cholesterol, and PEG-DMG (ratios shown in Table 10) in absolute ethanol at a concentration of about 15 mg/mL. The nitrogen to phosphate ratio is approximate to 3: 1.

[0365] Equal volume of siNA/carrier and lipid solutions are delivered with two FPLC pumps at the same flow rates to a mixing T connector. A back pressure valve is used to adjust to the desired particle size. The resulting milky mixture is collected in a sterile glass bottle. This mixture is then diluted slowly with an equal volume of citrate buffer, and filtered through an ion- exchange membrane to remove any free siNA/carrier in the mixture. Ultra filtration against citrate buffer (pH 4.0) is employed to remove ethanol (test stick from ALCO screen), and against PBS (pH 7.4) to exchange buffer. The final LNP is obtained by concentrating to a desired volume and sterile filtered through a 0.2 μιυ filter. The obtained LNPs are characterized in term of particle size, Zeta potential, alcohol content, total lipid content, nucleic acid encapsulated, and total nucleic acid concentration.

LNP Manufacture Process

[0366] In a non-limiting example, a LNP-086 siNA/carrier formulation is prepared in bulk as follows. The process consists of (1 ) preparing a lipid solution; (2) preparing an siNA/carrier solution; (3) mixing/particle formation; (4) incubation; (5) dilution; (6) ultrafiltration and concentration.

1 , Preparation of Lipid Solution

[0367] A 3-necked 2L round bottom flask, a condenser, measuring cylinders, and two 10L conical glass vessels are depyrogenated. The lipids are wanned to room temperature. Into the3- necked round bottom flask is transferred 50.44g of CLinDMA with a pipette and 43.32g of DSPC, 5.32g of Cholesterol , 6.96g of PEG-DMG, and 2.64g of linoleyl alcohol are added. To the mixture is added 1L of ethanol. The round bottom flask is placed in a heating mantle that is connected to a J-CHEM process controller. The lipid suspension is stirred under Argon with a stir bar and a condenser on top. A thermocouple probe is put into the suspension through one neck of the round bottom flask with a sealed adapter. The suspension is heated at 30 °C until it became clear. The solution is allowed to cool to room temperature and transferred to a conical glass vessel and sealed with a cap.

2. Preparation of siNA/Carrier Solution

[0368] Into a sterile container, such as the Coming storage bottle, is weighed 3.6 g times the water correction factor (approximately 1.2) of siNA-1 powder. The siNA is transferred to a depyrogenated 5 L glass vessel. The weighing container is rinsed 3x with citrate buffer (25mM, pH 4.0, and lOOmM Nad) and the rinses are placed into the 5 L vessel, QS with citrate buffer to 4 L. The concentration of the siNA solution is determined with a UV spectrometer using the following procedure. 20 is removed from the solution, diluted 50 times to 1000

and the UV reading recorded at A260 nm after blanking with citrate buffer. This is repeated. If the readings for the two samples are consistent, an average is taken and the concentration is calculated based on the extinction coefficients of the siNAs. If the final concentration are out of the range of 0.90 ± 0.01 mg/mL, the concentration is adjusted by adding more siNA/carrier powder, or adding more citrate buffer. This process is repeated for the second siNA, siNA-2.. Into a depyrogenated 10L glass vessel, 4 L of each 0.9 mg/mL siNA solution is transferred.

[0369] Alternatively, if the siNA/carrier solution comprised a single siNA duplex and or carrier instead of a cocktail of two or more siNA duplexes and/or carriers, then the siNA/carrier is dissolved in 25 mM citrate buffer (pH 4.0, 100 mM of NaCl) to give a final concentration of

[0370] The lipid/ethanol solution is then sterile/filtered through a Pall Acropak 20 0.8/0.2 μιτι sterile filter PN 12203 into a depyrogenated glass vessel using a Master Flex Peristaltic Pump Model 7520-40 to provide a sterile starting material for the encapsulation process. The filtration process is run at an 80 mL scale with a membrane area of 20 cm². The flow rate is 280 mL/minute. This process is scaleable by increasing the tubing diameter and the filtration area.

3. Particle formation - Mixing step

[0371] An A TA P900 pump is turned on and sanitized by placing 1000 mL of 1 N NaOH into a 1 L glass vessel and 1000 mL of 70% ethanol into a 1 L glass vessel and attaching the pump with a pressure lid to each vessel. A 2000 mL glass vessel is placed below the pump outlet and the flow rate is set to 40 mL/min for a 40 minute time period with argon flushing the system at 10 psi. When the sanitation is complete, the gas is turned off and the pump is stored in the solutions until ready for use. Prior to use, the pump flow is verified by using 200 mL of ethanol and 200 mL of sterile citrate buffer.

[0372] To the AKTA pump is attached the sterile lipid/ethanol solution, the sterile siNA/carrier or siNA/carrier cocktail /citrate buffer solution and a depyrogenated receiving vessel (2x batch size) with lid. The gas is turned on and the pressure maintained between 5 to 10 psi during mixing.

4. Incubation

[0373] The solution is held after mixing for a 22 ± 2 hour incubation. The incubation is done at room temperature (20 - 25°C) and the in-process solution was protected from light.

5. Dilution

[0374] The lipid siNA solution is diluted with an equal volume of Citrate buffer using a dual head peristaltic pump, Master Flex Peristaltic Pump, Model 7520-40 that is set up with equal lengths of tubing and a Tee connection and a flow rate of 360 mL/minute. 6. Ultrafiltration and Concentration

[0375] The ultrafiltration process is a timed process and the flow rates must be monitored carefully. This is a two step process; the first is a concentration step taking the diluted material from 32 liters to 3600 mLs and to a concentration of approximately 2 mg/mL.

[0376] In the first step, a Flexstand with a ultrafiltration membrane GE FN UFP-100-C-35A installed is attached to the quatroflow pump. 200 mL of WFI is added to the reservoir followed by 3 liters of 0.5 N sodium hydroxide which is then flushed through the retentate to waste. This process is repeated three times. Then 3 L WFI are flushed through the system twice followed by 3 L of citrate buffer. The pump is then drained.

[0377] The diluted LNP solution is placed into the reservoir to the 4 liter mark. The pump is turned on and the pump speed adjusted so the permeate flow rate is 300 mL/minute and the liquid level is constant at 4L in the reservoir. The pump is stopped when all the diluted LNP solution has been transferred to the reservoir. The diluted LNP solution is concentrated to 3600 mL in 240 minutes by adjusting the pump speed as necessary.

[0378] The second step is a diafiltration step exchanging the ethanol citrate buffer to phosphate buffered saline. The diafiltration step takes 3 hours and again the flow rates must be carefully monitored. During this step, the ethanol concentration is monitored by head space GC. After 3 hours (20 diafiltration volumes), a second concentration is undertaken to concentrate the solution to approximately 6 mg/mL or a volume of 1.2 liters. This material is collected into a depyrogenated glass vessel. The system is rinsed with 400 mL of PBS at high flow rate and the permeate line closed. This material is collected and added to the first collection. The expected concentration at this point is 4.5 mg/mL. The concentration and volume are determined.

[0379] The feed tubing of the peristaltic pump is placed into a container containing 72 L of PBS (0.05 μιη filtered) and the flow rate is adjusted initially to maintain a constant volume of 3600 mL in the reservoir and then increased to 400 mL/minute. The LNP solution is diafiltered with PBS (20 volumes) for 180 minutes. [0380] The LNP solution is concentrated to the 1.2 liter mark and collected into a depyrogenated 2 L graduated cylinder. 400 mL of PBS are added to the reservoir and the pump is recirculated for 2 minutes. The rinse is collected and added to the collected LNP solution in the graduated cylinder.

[0381] The obtained LNPs are characterized in terms of particle size, Zeta potential, alcohol content, total lipid content, nucleic acid encapsulated, and total nucleic acid concentration.

[0382] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein, as presently representative of preferred embodiments, are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

Table 7: MAPKl Accession Numbers

NM_002745 - SEQ ID NO: 1463

Homo sapiens, mitogen-activated protein kinase 1 (MAPKl ), transcript variant 1 , mRNA. NM_002745.4 GI:75709178

XM_001089660

Macaca mulatta mitogen-activated protein kinase 1 (MAPK l), mRNA

XM_001089600.2 GI:297260644

NM_01 1949

MUS musculus mitogen-activated protein kinase 1 (MAPKl) transcript variant 1 , mRNA. NMJ)1 1949.3 GI:8457991 1

Table 8

Non-limiting examples of Stabilization Chemistries for chemically modified siNA constructs

Chemistry pyrimidine purine caps p=S Strand

"Stab 00" Ribo Ribo TT at 3'- S/AS ends

"Stab 1" Ribo Ribo - 5 at 5 '-end S/AS

1 at 3 '-end

"Stab 2" Ribo Ribo - All linkages Usually AS

"Stab 3" 2'-fluoro Ribo - 4 at 5 '-end Usually S

4 at 3 '-end

"Stab 4" 2'-fluoro Ribo 5' and 3'- - Usually S ends

"Stab 5" 2'-fluoiO Ribo - 1 at 3 '-end Usually AS

"Stab 6" 2'-0-Methyl Ribo 5' and 3'- - Usually S ends

"Stab 7" 2'-fluoro 2'-deoxy 5' and 3'- - Usually S ends

"Stab 8" 2'-fluoro 2'-0- - 1 at 3 '-end S/AS

Methyl

"Stab 9" Ribo Ribo 5' and 3'- - Usually S ends

"Stab 10" Ribo Ribo - 1 at 3 '-end Usually AS

"Stab 11" 2'-fluoro 2'-deoxy - 1 at 3 '-end Usually AS

"Stab 12" 2'-fluoro LNA 5 ' and 3'- Usually S ends

"Stab 13" 2ⁱ-fIuoro LNA 1 at 3 '-end Usually AS

"Stab 14" 2'-fluoro 2'-deoxy 2 at 5 '-end Usually AS

1 at 3 '-end

"Stab 15" 2'-deoxy 2'-deoxy 2 at 5 '-end Usually AS

1 at 3 '-end

"Stab 16" Ribo 2'-0- 5' and 3'- Usually S

Methyl ends

"Stab 17" 2'-0-Methyl 2'-0- 5' and 3'- Usually S

Methyl ends

"Stab 18" 2'-fluoro 2'-0- 5' and 3'- Usually S

Methyl ends

"Stab 19" 2'-fluoro 2'-0- 3'-end S/AS

Methyl

"Stab 20" 2'-fluoro 2'-deoxy 3' -end Usually AS

"Stab 21" 2'-fluoro Ribo 3 '-end Usually AS

"Stab 22" Ribo Ribo 3 '-end Usually AS

"Stab 23" 2'-fluoro* 2'-deoxy* 5 ' and 3'- Usually S ends

"Stab 24" 2'-fluoro* 2'-0- - 1 at 3' -end S/AS

Methyl*

"Stab 25" 2'-fluoro* 2'-0- - 1 at 3 '-end S/AS

Methyl*

"Stab 26" 2'-fluoro* T-O- - S/AS

Methyl*

"Stab 27" 2'-fluoro* 2'-0- 3 '-end S/AS

Methyl*

"Stab 28" 2'-fluoro* 2'-0- 3 '-end S/AS

Methyl*

"Stab 29" 2'-fluoro* 2'-0- 1 at 3 '-end S/AS

Methyl*

"Stab 30" 2'-fluoro* 2'-0- S/AS

Methyl*

"Stab 31" 2'-iluoro* 2'-0- 3 '-end S/AS

Methyl*

"Stab 32" 2'-fluoro 2'-0- S/AS

Methyl

"Stab 33" 2'-fluoro 2'-deoxy* 5' and 3'- - Usually S ends

"Stab 34" 2'-fluoro 2^!-0- 5' and 3'- Usually S

Methyl* ends

"Stab 35" 2'-fluoro*† 2'-0- Usually AS

Methyl*†

"Stab 36" 2'-fluoro*† 2'-0- Usually AS

Methyl*†

"Stab04H" 2'-fluoro RiboJ 5' and 3'- 1 at 3 '-end Ususally S ends

"Stab06C" 2'-0-MethylJ Ribo$ 5' and 3'- Ususally S ends

"Stab07H" 2'-fluoro$ 2'-deoxyJ 5' and 3'- 1 at 3 '-end Ususally S ends

"Stab07mU" 2'-fluoroJ 2'-deoxy$ 5' and 3'- Ususally S ends

"Stab09H" Ribo$ Ribo 5' and 3'- 1 at 3 '-end Ususally S ends

"Stabl6C" RiboJ 2'-0- 5' and 3'- Ususally S

MethylJ ends

"Stabl6H" RiboJ 2'-0- 5' and 3'- 1 at 3'-end Ususally S

MethylJ ends

"Stabl8C" 2'-fluoroJ r-o- 5' and 3'- Ususally S

MethylJ ends

"Stabl8H" 2'-fluoiO$ r-o- 5' and 3'- 1 at 3 '-end Ususally S

MethylJ ends

"Stab52H" 2'-0-Methyli Ribo$ 5' and 3'- 1 at 3 '-end Ususally S ends

"Stab05C" 2'-fluoro RiboJ Ususally AS

"Stab05N" 2'-fluoroJ RiboJ 1 at 3 '-end Ususally AS

"StablOC RiboJ RiboJ Ususally AS

"StablON" RiboJ RiboJ 1 at 3 '-end Ususally AS

"Stab35G*" 2'-fluoro$ 2'-0- Ususally AS

Methyl

"Stab35N*" 2'-fluoro| 2'-0- 1 at 3 '-end Ususally AS

MethylJ

"Stab35rev*" 2'-0-MethylJ 2'-fluoroJ Ususally AS

"Stab50*" RiboJ 2'-0- Ususally AS

MethylJ

"Stab53*" 2'-0-Methyli RiboJ Ususally AS

"Stab53N*" 2'-0-Methyli RiboJ 1 at 3 '-end Ususally AS

Stab54 RiboJ 2'-fluoro Ususally AS

CAP = any terminal cap, see for example Figure 5.

All Stab chemistries can be used in combination with each other for duplexes of the invention (e.g., as combinations of sense and antisense strand chemistries), or alternately can be used in isolation, e.g., for single stranded nucleic acid molecules of the invention.

All Stab chemistries can comprise 3'-overhang nucleotides having 2'-0-alkyl, 2'-deoxy-2'-fluoro, 2'-deoxy, LNA or other modified nucleotides or non-nucleotides.

All Stab chemistries typically comprise about 19-21 nucleotides, but can vary as described herein.

All Stab chemistries can also include a single ribonucleotide in the sense or passenger strand at the 11 ^th base paired position of the double-stranded nucleic acid duplex as determined from the 5'-end of the antisense or guide strand (see Figure 4C).

S = sense strand.

AS = antisense strand

*Stab 23 has a single ribonucleotide adjacent to 3 '-CAP.

*Stab 24 and Stab 28 have a single ribonucleotide at 5'-tenninus.

*Stab 25, Stab 26, Stab 27, Stab 35, Stab 35G*, Stab 35N*, Stab 35rev*, Stab 36, Stab 50*,

Stab53*, Stab 53N*, and Stab 54 have three ribonucleotides at 5 '-terminus.

*Stab 29, Stab 30, Stab 31 , Stab 33, and Stab 34 any purine at first three nucleotide positions from 5'-terminus are ribonucleotides.

p = phosphorothioate linkage.

†Stab 35 has 2'-0-methyl U at 3'-overhangs and three ribonucleotides at 5'-terminus.

†Stab 36 has 2'-0-methyl overhangs that are complementary to the target sequence, (naturally occurring overhangs) and three ribonucleotides at 5'-terminus.

J - Stab 04H, Stab 06C, Stabl07H, Stab07mU, Stab09H, Stabl6C, Stab 16H, Stabl8C, Stab 18H, Stab 52H, Stab 05C, StabOSN, StablOC, StablON, Stab35G*, Stab35N*, Stab35N*, Stab35rev*, Stab 50*, Stab 53*, Stab 53N*, Stab 54 have two 2'-0-methyl U 3'-overhangs.

Stab35G*, Stab 35N*, Stab35rev* Stab50*, Stab53*, and Stab53N* do not allow for a 2'-0- methyl modification at position 14 of the guide strand as determined from the 5 '-end. Table 9

A. 2.5 μηιοΐ Synthesis Cycle ABl 394 Instrument

B. 0.2 μπιοΐ Synthesis Cycle ABl 394 Instrument

C. 0.2 μηιοΐ Synthesis Cycle 96 well Instrument

• Wait time does not include contact time during delivery.

• Tandem synthesis utilizes double coupling of linker molecule

Table 10

Lipid Nanoparticle (LNP) Formulations

of 2

L109 DMOBA/DSPC/Cholesterol/2 PEG-Chol, 50 / 20/ 28 / 2

N/P ratio of 2

L110 DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3

ratio of 1.5

LI 11 DMOBA/Cholesterol/2KPEG-DMG, N/P 67/30/3

ratio of 1.5

LI 12 DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 52/45/3

of 1.5

LI 13 DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 67 /30 / 3

of 1.5

L114 DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3

ratio of 2

L115 DMOBACholesterol/2KPEG-DMG, N/P 67/30/3

ratio of 2

L116 DMLBA/Cholesterol/2 PEG-DMG, N/P ratio 52/45/3

of2

L117 DMLBA/Cholesterol/2 PEG-DMG, N/P ratio 52/45/3

of 2

L118 LinCDMA/DSPC/Cholesterol/2KPEG- 43/38/ 10/2/7

DMG/Linoleyl alcohol, N/P ratio of 2.85

L121 2-CLlM/DSPC/Cholesterol/2KPEG-DMG/, 48/40/ 10/2

N/P ratio of 3

L122 2-CLIM/ Cholesterol/2KPEG-DMG/, N/P 68/30/2

ratio of 3

L123 CLinDMA/DSPC/Cholesterol/2KPEG- 43/37/ 10/3/7

DMG/Linoleyl alcohol, N/P ratio of 2.85

L124 CLinDMA/DSPC/Cholesterol/2KPEG- 43 /36/ 10/4/7

DMG/Linoleyl alcohol, N/P ratio of 2.85

L130 CLinDMA / DOPC / Choi / PEG-n-DMG, 48/39/10/3

N/P ratio of 3

L131 DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 52/43/5

of 3

L132 DMOBA/Cholesterol/2KPEG-DMG, N/P 52/43/5

ratio of 3

L133 CLinDMA / DOPC / Choi / PEG-n-DMG, 48/40/10/2

N/P ratio of 3

L134 CLinDMA / DOPC / Choi / PEG-n-DMG, 48/37/10/5

N/P ratio of 3

L149 COIM/DSPC/Cholesterol/2KPEG-DMG/, N/P 48/40/ 10/2 ratio of 3

L155 CLinDMA/DOPC/Cholesterol/2KPEG- 43/38/ 10/2/7

DMG/Linoleyl alcohol, N/P ratio of 2.85

L156 CLinDMA/DOPC/Cholesterol/2 PEG-DMG, 45/43 / 10/2

N/P ratio of 2.85

LI 62 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/43/ 10/2 N/P ratio of 2.5

L163 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/43/ 10/2

N/P ratio of 2

L164 CLinDMA/DOPC/Cholesterol/2 PEG-DMG, 45/43/ 10/2

NP ratio of 2.25

L165 CLinDMA/DOPC/Cholesterol/2 PEG-DMG, 40/43/ 15/2

N/P ratio of 2.25

LI 66 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/43/ 15/2

N/P ratio of 2.5

L167 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/43/ 15/2

N/P ratio of 2

LI 74 CLinD A/DSPC/DOPC/Cholesterol/2KPEG- 43/9/27/ 10/4/7

DMG/Linoleyl alcohol, N/P ratio of 2.85

L175 CLinDMA/DSPC/DOPC/Cholesterol/2KPEG- 43/27/9/ 10/4/7

DMG/Linoleyl alcohol, N/P ratio of 2.85

LI 76 CLinDMA/DOPC/ChoIesteroI/2KPEG- 43/36/ 10/4/7

DMG/Linoleyl alcohol, N/P ratio of 2.85

L180 CLinDMA/DOPC/Cholesterol/2KPEG- 43/36/ 10/4/7

DMGLinoleyl alcohol, N/P ratio of 2.25

L181 CLinDMA/DOPC/Cholesterol/2KPEG- 43/36/10/4/7

DMGLinoleyl alcohol, N/P ratio of 2

LI 82 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/41 / 10/4

N/P ratio of 2.25

L197 CODMA/DOPC/Cholesterol/2KPEG-DMG, 43/36/ 10/4/7

N/P ratio of 2.85

L198 CLinDMA/DOPC/Cholesterol/2KPEG- 43/34/ 10/4/2/

DMG/2KPEG-DSG/L5noleyl alcohol, N/P 7 ratio of 2.85

L199 CLinDMA/DOPC/Cholesterol/2KPEG- 43/34/ 10/6/7

DMG/Linoleyl alcohol, N/P ratio of 2.85

L200 CLinDMA/Cholesterol/2 PEG-DMG, NP 50/46/4

ratio of 3.0

L201 CLinDMA/Cholesterol/2KPEG-DMG, N/P 50/44/6

ratio of 3.0

L206 CLinDMA/Cholesterol/2KPEG-DMG, N/P 40/56/4

ratio of 3.0

L207 CLinDMACholesterol/2KPEG-DMG, NP 60/36/4

ratio of 3.0

L208 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/10/46/4

N/P ratio of 3.0

L209 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 60/ 10/26/4

N/P ratio of 3.0

D000 DLinDMACholesterol/PEG-DMG/DSPC, 40/48/2/ 10

N/P ratio of 2.8

D001 DLinDMA/Cholesterol/S-PEG-C- 40/48/2/ 10

DMA/DSPC, N/P ratio of 6 N/P ratio - Nitrogen:Phosphorous ratio between cationic lipid and nucleic acid

The 2 PEG utilized is PEG2000, a polydispersion which can typically vary from -3000 Da (i.e., where PEG(n) is about 33 to about 67, or on average -45).

Table 11

CLinDMA structure

pCLinDMA structure

eCLinDMA structure

DEGCLinDMA structure

PEG-n-DMG structure

= about 33 to 67, average = 45 for 2KPEG/PEG2000 DMOBA structure

DMLBA structure

DOBA structure

DSPC structure

Cholesterol structure

2KPEG-CA olesterol structure

n = about 33 to 67, average = 45 for 2KPEG/PEG2000

2 PEG-DMG structure

n = about 33 to 67, average = 45 for 2KPEG/PEG2000

COIM STRUCTURE

-CLIM AND 2-CLIM STRUCTURE

171

Claims

What we claim is:

1 . An isolated double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of MAPK1 , wherein

(a) the siNA comprises a sense strand and an antisense strand;

(b) each strand is independently 15 to 30 nucleotides in length; and

(c) at least one strand comprises at least a 15 nucleotide sequence selected from the group consisting of SEQ ID NOS:l -1462.

2. A double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of MAPK1 , wherein

(a) the siNA comprises a sense strand and an antisense strand;

(b) each strand is independently 1 to 30 nucleotides in length; and

(c) the antisense strand comprises at least 15 nucleotides having sequence complementary to any of:

5'-AGAACAUCAUUGGAAUCAA-3' (SEQ ID NO: 1 );

5'-UAUCCAUUCAGCUAACGUU-3' (SEQ ID NO: 2);

5'-UCCAUUCAGCUAACGUUCU-3' (SEQ ID NO: 3); or

5'-UUAUGAUAAUGUCAACAAA-3' (SEQ ID NO: 4).

3. A double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of MAP 1 , wherein

(a) the siNA comprises a sense strand and an antisense strand;

(b) each strand is independently 15 to 30 nucleotides in length; and

(c) the antisense strand comprises at least a 1 5 nucleotide sequence of:

5'-UUGAUUCCAAUGAUGUUCU-3' (SEQ ID NO: 1210); 5'-AACGUUAGCUGAAUGGAUA-3' (SEQ ID NO: 121 1);

5- -AGAACGUUAGCUGAAUGGA-3' (SEQ ID NO: 1212); or

5^>-UUUGUUGACAUUAUCAUAA-3' (SEQ ID NO: 1213); and wherein one or more of the nucleotides are optionally chemically modified.

The double-stranded short interfering nucleic acid (siNA) molecule of claim 3, wherein the sense strand comprises at least a 15 nucleotide sequence of:

5'-AGAACAUCAUUGGAAUCAA-3' (SEQ ID NO: 1);

5'-UAUCCAUUCAGCUAACGUU-3' (SEQ ID NO: 2);

5'-UCCAUUCAGCUAACGUUCU-3' (SEQ ID NO: 3); or

5'-UUAUGAUAAUGUCAACAAA-3' (SEQ ID NO: 4); and wherein one or more of the nucleotides are optionally chemically modified.

A double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of MAPK1, wherein the siNA comprises any of:

5'-AGAACAUCAUUGGAAUCAA-3' (SEQ ID NO: 1 ) and 5'- UUGAUUCCAAUGAUGUUCU-3' (SEQ ID NO: 1210); or

5'-UAUCCAUUCAGCUAACGUU-3' (SEQ ID NO: 2); and 5'- A ACGUU AGCUG A AUGG AU A-3 ' (SEQ ID NO: 121 1); or

5'-UCCAUUCAGCUAACGUUCU-3' (SEQ ID NO: 3) and 5'- AGAACGUUAGCUGAAUGGA-3' (SEQ ID NO: 1212); or

5'-UUAUGAUAAUGUCAACAAA-3' (SEQ ID NO: 4); and 5'- UUUGUUGACAUUAUCAUAA-3' (SEQ ID NO: 1213).

The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1, 2, 3, or 5, wherein at least one nucleotide is a chemically modified nucleotide.

The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1 , 2, 3, or 5 further comprising at least one non-nucleotide.

8. The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1 , 2, 3, or 5, wherein at least one nucleotide comprises a universal base.

9. The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1 , 2, 3, or 5, having at least one phosphorothioate internucleotide linkage.

10. The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1 , 2, 3, or 5, comprising a cap on the 3'-end, 5'-end or both 3' and 5' ends of at least one strand.

1 1. The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1, 2, 3, or 5, comprising one or more 3'- overhang nucleotides on one or both strands.

12. The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1 , 2, 3, or 5, wherein the 5' end of the antisense strand is phosphorylated.

13. The double-stranded short interfering nucleic acid (siNA) molecule of claim 1 1 , wherein the 3'-overhang nucleotides on at least one strand are 2'-0-methyI nucleotides.

14. The double-stranded short interfering nucleic acid (siNA) molecule of claim 13, wherein the 2'-0-methyl nucleotides are linked with a phosphorothioate internucleotide linkage.

15. The double-stranded short interfering nucleic acid (siNA) molecule of claim 6, wherein the chemically modified nucleotide is a 2'-deoxy-2'-fluoro nucleotide.

36. The double-stranded short interfering nucleic acid (siNA) molecule of claim 6, wherein the chemically modified nucleotide is a 2'-deoxy nucleotide.

17. The double-stranded short interfering nucleic acid (siNA) molecule of claim 6, wherein the chemically modified nucleotide is a 2'-0-alkyl nucleotide.

18. The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1 , 2, 3, or 5, wherein five or more pyrimidine nucleotides in one or both strands are 2'- deoxy-2'-fluoro pyrimidine nucleotides.

19. The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1, 2, 3, or 5, wherein five or more pyrimidine nucleotides in one or both strands are 2'-0- methyl pyrimidine nucleotides.

20. The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1 , 2, 3, or 5, wherein five or more purine nucleotides in one or both strands are 2'-deoxy- 2'-fluoro purine nucleotides.

21 . The double-stranded short interfering nucleic acid (siNA) molecule according to claims 1 , 2, or 4, wherein five or more purine nucleotides in one or both strands are 2'-0- methyl purine nucleotides.

22. The double-stranded short interfering nucleic acid (siNA) molecule of claim 18, wherein five or more purine nucleotides in one or both strands are 2'-0-methyl purine nucleotides.

23. The double-stranded short interfering nucleic acid (siNA) molecule of claim 19, wherein five or more purine nucleotides in one or both strands are 2'-deoxy-2'-fluoro nucleotides. 24. A double-stranded short interfering nucleic acid (siNA) molecule, comprising formula A) having a sense strand and an antisense strand:

(A) wherein, the upper strand is the sense strand and the lower strand is the antisense strand of the double-stranded nucleic acid molecule; wherein the antisense strand comprises at least a 15 nucleotide sequence of SEQ ID NO: 1210, SEQ ID NO: 1211 , SEQ ID NO: 1212, or SEQ ID NO: 1213, and the sense strand comprises a sequence having complementarity to the antisense strand; each N is independently a nucleotide which is unmodified or chemically modified or a non-nucleotide; each B is a terminal cap that is present or absent; (N) represents overhanging nucleotides, each of which is independently unmodified or chemically modified;

X5 is an integer from 0 to 6, provided that the sum of X4 and X5 is 15-30.

The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein

(a) one or more pyrimidine nucleotides in Νχ₄ positions are independently 2'-deoxy- 2'-fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof;

(b) one or more purine nucleotides in Νχ₄ positions are independently 2'-deoxy-2'- fluoro nucleotides, 2'-0-a!kyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof;

(c) one or more pyrimidine nucleotides in Νχ₃ positions are independently 2'-deoxy- 2'-fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof; and

(d) one or more purine nucleotides in χ₃ positions are independently 2'-deoxy-2'- fluoro nucleotides, 2'-0-alkyl nucleotides, 2'-deoxy nucleotides, ribonucleotides, or any combination thereof.

The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein X5 is 3.

The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein X5 is 0.

28. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein XI is 2 and X2 is 2.

29. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein X5 is 0, XI is 2 and X2 is 2.

30. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein X5 is 3, XI is 2 and X2 is 2.

31. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein one or more nucleotides has an universal base.

32. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein the nucleotide at position 14 from the 5'-end of the antisense strand is not a 2'- O-methyl nucleotide.

33. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein one or more overhang nucleotides is a 2'-0-methyl nucleotide.

34. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, comprising at least one phosphorothioate internucleotide linkage.

35. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 22, wherein X5 = 0, 1, 2, or 3; each XI and X2 = 1 or 2; X3 = 18, 19, 20, 21 , 22, or 24, and X4 = 17, 18, 19, 20, 21 , 22, or 23.

36. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 24, wherein X5 = 3; each XI and X2 = 2; X3 = 19, and X4 = 16.

37. The double-stranded short interfering nucleic acid (siNA) molecule according to claim

24, wherein X5 - 0; each XI and X2 = 2; X3 = 19, and X4 = 19.

38. The double-stranded short interfering nucleic acid (siNA) molecule according to claim

25, wherein

(a) 1 , 2, 3, 4, 5 or more pyrimidine nucleotides in Νχ₄ positions are 2'-deoxy-2'- fluoro nucleotides; (b) 1 , 2, 3, 4, 5 or more purine nucleotides in Νχ₄ positions are 2'-0-alkyl nucleotides;

(c) 1 , 2. 3, 4, 5 or more pyrimidine nucleotides in Νχ₃ positions are 2'-deoxy-2'- fluoro nucleotides; and

(d) 1 , 2, 3, 4, 5 or more purine nucleotides in Νχ₃ positions are 2'-deoxy nucleotides.

39. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 25, wherein

(a) 1 , 2, 3, 4, 5 or more pyrimidine nucleotides in Νχ₄ positions are 2'-0-alkyl nucleotides;

(b) 1, 2, 3, 4, 5 or more purine nucleotides in Νχ₄ positions are ribonucleotides;

(c) 1 , 2, 3, 4, 5 or more pyrimidine nucleotides in Νχ₃ positions are 2'-0-alkyl nucleotides; and

(d) 1, 2, 3, 4, 5 or more purine nucleotides in Νχ₃ positions are ribonucleotides.

40. The double-stranded short interfering nucleic acid (siNA) molecule according to claim 25, wherein

(a) 1 , 2, 3. 4, 5 or more pyrimidine nucleotides in Νχ₄ positions are 2'-deoxy-2'- fluoro nucleotides;

(d) 1 , 2, 3, 4, 5 or more purine nucleotides in Νχ positions are 2'-deoxy-2'-fluoro nucleotides.

41. The double-stranded short interfering nucleic acid (siNA) molecule according to any of claims 38, 39 or 40, comprising one or more phosphorothioate internucleotide linkages.

42. A composition comprising the double-stranded short interfering nucleic acid (siNA) of any of claims 1, 2, 3, 5, or 24 in a pharmaceutically acceptable carrier or diluent.

43. A method of treating a human subject suffering from a condition which is mediated by the action, or by loss of action, of MAPKl, which comprises administering to said subject an effective amount of the double-stranded short interfering nucleic acid (siNA) molecule of claim 1.

44. The method according to claim 1 , wherein the condition is cancer.

45. A method of treating a human subject suffering from a condition which is mediated by the action, or by loss of action, of MAPKl , which comprises administering to said subject an effective amount of the double-stranded short interfering nucleic acid (siNA) molecule of claim 3.

46. The method according to claim 45, wherein the condition is cancer.