US20090023676A1 - RNA Interference Mediated Inhibition of MAP Kinase Gene Expression or Expression of Genes Involved in MAP Kinase Pathway Using Short Interfering Nucleic Acid (SiNA) - Google Patents

Abstract

The present invention concerns methods and reagents useful in modulating MAP kinase gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to 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 capable of mediating RNA interference (RNAi) against c-JUN, JNK, p38, and ERK gene expression, useful in the treatment of cancer, inflammation, obesity and insulin resistance (e.g. Type I and Type II diabetes).

Description

• This application is a continuation of U.S. patent application Ser. No. 10/424,339, filed Apr. 25, 2003, which is a continuation-in-part of International Patent Application No. PCT/US03/02510, filed Jan. 28, 2003, and is a continuation-in-part of International Patent Application No. PCT/US03/05346, filed Feb. 20, 2003, and is a continuation-in-part of International Patent Application No. PCT/US03/05028, filed Feb. 20, 2003, which each claim the benefit of U.S. Provisional Application No. 60/358,580 filed Feb. 20, 2002, U.S. Provisional Application No. 60/363,124 filed Mar. 11, 2002, U.S. Provisional Application No. 60/386,782 filed Jun. 6, 2002, U.S. Provisional Application No. 60/406,784 filed Aug. 29, 2002, U.S. Provisional Application No. 60/408,378 filed Sep. 5, 2002, U.S. Provisional Application No. 60/409,293 filed Sep. 9, 2002, and U.S. Provisional Application No. 60/440,129 filed Jan. 15, 2003. The instant application claims the benefit of all the listed applications, which are hereby incorporated by reference herein in their entireties, including the drawings.
SEQUENCE LISTING
• 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 “SequenceListing45USCNT”, created on Aug. 27, 2008, which is 468,031 bytes in size.
FIELD OF THE INVENTION
• The present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases that respond to the modulation of mitogen activated protein kinase (MAP kinase) gene expression and/or activity. The present invention also concerns compounds, compositions, methods relating to the modulation of expression or activity of genes involved in the MAP kinase pathway. Specifically, the invention relates to 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 capable of mediating RNA interference (RNAi) against genes involved in the Jun amino-terminal kinase (JNK), p38, and/or ERK pathway, such as c-JUN. More specifically, the invention relates to 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 capable of mediating RNA interference (RNAi) against Jun amino-terminal kinase (JNK), p38, ERK, and/or c-JUN genes.
BACKGROUND OF THE INVENTION
• The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.
• RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998,Nature,391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., 1999,Trends Genet.,15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
• The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001,Nature,409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir et al., 2001,Genes Dev.,15, 188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001,Science,293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001,Genes Dev.,15, 188).
• RNAi has been studied in a variety of systems. Fire et al., 1998,Nature,391, 806, were the first to observe RNAi inC. elegans. Wianny and Goetz, 1999,Nature Cell Biol.,2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,Nature,404, 293, describe RNAi inDrosophilacells transfected with dsRNA. Elbashir et al., 2001,Nature,411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work inDrosophilaembryonic lysates (Elbashir et al., 2001,EMBO J.,20, 6877) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the guide sequence (Elbashir et al., 2001,EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell,107, 309).
• Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001,EMBO J.,20, 6877). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA. Kreutzer et al., Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in siRNA molecules.
• Parrish et al., 2000,Molecular Cell,6, 1977-1087, tested certain chemical modifications targeting the unc-22 gene inC. elegansusing long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi. Further, Parrish et al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081. The authors also tested certain modifications at the 2′-position of the nucleotide sugar in the long siRNA transcripts and found that substituting deoxynucleotides for ribonucleotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil substitution appeared to be tolerated, Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.
• The use of longer dsRNA has been described. For example, Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describe aDrosophilain vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001,Chem. Biochem.,2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger of activating interferon response. Li et al., International PCT Publication No. WO 00/44914, describe the use of specific dsRNAs for attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for introducing certain dsRNA molecules into cells for use in inhibiting gene expression. Plaetinck et al., International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al., International PCT Publication No. 99/53050, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844, describe specific DNA constructs for use in facilitating gene silencing in targeted organisms.
• Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al., 2000,Molecular Cell,6, 1977-1087, describe specific chemically-modified siRNA constructs targeting the unc-22 gene ofC. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al., International PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475, describe certain methods for isolating aNeurosporasilencing gene and uses thereof. Reed et al., International PCT Publication No. WO 01/68836, describe certain methods for gene silencing in plants. Honer et al., International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certainDrosophila-derived gene products that may be related to RNAi inDrosophila. Arndt et al., International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain dsRNAs. Echeverri et al., International PCT Publication No. WO 02/38805, describe certainC. elegansgenes identified via RNAi. Kreutzer et al., International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certain methods for inhibiting gene expression using RNAi. Graham et al., International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (greater than 25 nucleotide) constructs that mediate RNAi.
SUMMARY OF THE INVENTION
• This invention relates to compounds, compositions, and methods useful for modulating the expression of genes associated with mitogen activated protein kinase (MAP kinase) gene expression pathways (see for exampleFIG. 12) by RNA interference (RNAi) using short interfering nucleic acid (siNA) molecules. In particular, the instant invention features 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 and methods used to modulate the expression of MAP kinase genes, including c-JUN, JNK genes such as JNK1 and JNK2, ERK genes such as ERK1 and ERK2, and p38 genes. A siNA of the invention can be unmodified or chemically-modified. A siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating telomerase gene expression or activity in cells by RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA having multiple chemical modifications retains its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.
• In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding MAP kinase proteins, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as MAP kinases. The description below of the various aspects and embodiments of the invention is provided with reference to exemplary MAP kinases such as JNK1 (also referred to as MAPK8, for example Genbank Accession No. NM_—002750), p38 (also referred to as MAPK14, for example Genbank Accession No. NM_—139012), ERK2 (also referred to as MAPK1, for example Genbank Accession No. NM_—002745), and ERK1 (also referred to as MAPK3, for example Genbank Accession XM_—055766) genes. However, the various aspects and embodiments are also directed to other MAP kinases referred to by Accession number in Table 1 and other genes involved in MAP kinase pathways such those genes encoding c-JUN (for example Genbank Accession No. NM_—002228), TNF-alpha (for example Genbank Accession No. M10988), interleukins such as IL-8 (for example Genbank Accession No. M68932), and activating proteins such as AP-1 (for example Genbank Accession No. NM_—013277). The various aspects and embodiments are also directed to other genes that are involved in the MAP kinase pathways of gene expression. Those additional genes can be analyzed for target sites using the methods described for MAP kinase genes herein. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.
• In one embodiment, the invention features a siNA molecule that down-regulates expression of a MAP kinase gene, for example, wherein the MAP kinase gene comprises MAP kinase encoding sequence (e.g., c-JUN, JNK1, JNK2, p38, ERK1, or ERK2).
• In one embodiment, the invention features a siNA molecule having RNAi activity against MAP kinase RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having MAP kinase encoding sequence, such as those sequences having MAP kinase GenBank Accession Nos. shown in Table I. In another embodiment, the invention features a siNA molecule having RNAi activity against MAP kinase RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having other MAP kinase encoding sequence, such as mutant MAP kinase genes, splice variants of MAP kinase genes, and other MAP kinase ligands and receptors. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention.
• In another embodiment, the invention features a siNA molecule having RNAi activity against a MAP kinase gene, wherein the siNA molecule comprises nucleotide sequence complementary to nucleotide sequence of a MAP kinase gene, such as those MAP kinase sequences having GenBank Accession Nos. shown in Table I or other MAP kinase encoding sequence, such as mutant MAP kinase genes, splice variants of MAP kinase genes, and other MAP kinase ligands and receptors. In another embodiment, a siNA molecule of the invention includes nucleotide sequence that can interact with nucleotide sequence of a MAP kinase gene and thereby mediate silencing of MAP kinase gene expression, for example, wherein the siNA mediates regulation of MAP kinase gene expression by cellular processes that modulate the chromatin structure of the MAP kinase gene and prevent transcription of the MAP kinase gene.
• In another embodiment, the invention features a siNA molecule comprising nucleotide sequence, for example, nucleotide sequence in the antisense region of the siNA molecule, that is complementary to a nucleotide sequence or portion of sequence of a MAP kinase gene. In another embodiment, the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a MAP kinase gene sequence or a portion thereof.
• In one embodiment, the antisense region of ERK2 siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 1-163, or 1113-1116. The antisense region can also comprise sequence having any of SEQ ID NOs. 164-326, 1133-1136, 1141-1144, or 1149-1152. In another embodiment, the sense region of ERK2 siNA constructs can comprise sequence having any of SEQ ID NOs. 1-163, 1113-1116, 1129-1132, 1137-1140, or 1145-1148.
• In one embodiment, the antisense region of ERK1 siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 327-431, or 1117-1120. The antisense region can also comprise sequence having any of SEQ ID NOs. 432-536, 1157-1160, 1165-1168, or 1173-1176. In another embodiment, the sense region of ERK1 siNA constructs can comprise sequence having any of SEQ ID NOs. 327-431, 1117-1120, 1153-1156, 1161-1164, or 1169-1172.
• In one embodiment, the antisense region of JNK1 siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 537-615 or 1121-1124. The antisense region can also comprise sequence having any of SEQ ID NOs. 616-694, 1181-1184, 1189-1192, 1197-1200, 1237, 1239, 1241, 1243, 1245, or 1246. In another embodiment, the sense region of JNK1 constructs can comprise sequence having any of SEQ ID NOs. 537-615, 1121-1124, 1177-1180, 1185-1188, 1193-1196, 1236, 1238, 1240, 1242, or 1244. The sense region can comprise a sequence of SEQ ID NO. 1225 and the antisense region can comprise a sequence of SEQ ID NO. 1226. The sense region can comprise a sequence of SEQ ID NO. 1227 and the antisense region can comprise a sequence of SEQ ID NO. 1228. The sense region can comprise a sequence of SEQ ID NO. 1229 and the antisense region can comprise a sequence of SEQ ID NO. 1230. The sense region can comprise a sequence of SEQ ID NO. 1231 and the antisense region can comprise a sequence of SEQ ID NO. 1232. The sense region can comprise a sequence of SEQ ID NO. 1233 and the antisense region can comprise a sequence of SEQ ID NO. 1234. The sense region can comprise a sequence of SEQ ID NO. 1231 and the antisense region can comprise a sequence of SEQ ID NO. 1235.
• In one embodiment, the antisense region of p38 siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 695-903 or 1125-1128. The antisense region can also comprise sequence having any of SEQ ID NOs. 904-1112, 1205-1208, 1213-1216, or 1221-1224. In another embodiment, the sense region of p38 siNA constructs can comprise sequence having any of SEQ ID NOs. 695-903, 1125-1128, 1201-1204, 1209-1212, or 1217-1220.
• In one embodiment, the antisense region of c-JUN siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 1247-1427 or 1609-1616. In one embodiment, the antisense region of c-JUN siNA constructs can comprise sequence having any of SEQ ID NOs. 1428-1608, 1625-1632, 1641-1648, 1657-1664, 1673-1680, 1698, 1700, 1702, 1705, 1707, 1709, 1711, or 1714. In another embodiment, the sense region of c-JUN siNA constructs can comprise sequence having any of SEQ ID NOs. 1247-1427, 1609-1616, 1617-1624, 1633-1640, 1649-1656, 1665-1672, 1697, 1699, 1701, 1703, 1704, 1706, 1708, 1710, 1712, or 1713.
• In one embodiment, a siNA molecule of the invention comprises any of SEQ ID NOs. 1-1714. The sequences shown in SEQ ID NOs: 1-1714 are not limiting. A siNA molecule of the invention can comprise any contiguous MAP kinase sequence (e.g., about 19 to about 25, or about 19, 20, 21, 22, 23, 24 or 25 contiguous MAP kinase nucleotides).
• In yet another embodiment, the invention features a siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I. Chemical modifications in Tables III and IV and described herein can be applied to any siRNA construct of the invention.
• In one embodiment of the invention a siNA molecule comprises an antisense strand having about 19 to about 29 nucleotides, wherein the antisense strand is complementary to a RNA sequence encoding a MAP kinase protein, and wherein said siNA further comprises a sense strand having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences with at least about 19 complementary nucleotides.
• In another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, wherein the antisense region is complementary to a RNA sequence encoding a MAP kinase protein, and wherein said siNA further comprises a sense region having about 19 to about 29 nucleotides, wherein said sense region and said antisense region comprise a linear molecule with at least about 19 complementary nucleotides.
• In one embodiment of the invention a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a MAP kinase protein or a portion thereof. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a MAP kinase gene or a portion thereof.
• In another embodiment, a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a MAP kinase protein or a portion thereof. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a MAP kinase gene or a portion thereof.
• In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a MAP kinase gene. Because MAP kinase genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of MAP kinase genes (and associated receptor or ligand genes) or alternately specific MAP kinase genes by selecting sequences that are either shared amongst different MAP kinase targets or alternatively that are unique for a specific MAP kinase target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of MAP kinase RNA sequence having homology between several MAP kinase genes so as to target several MAP kinase genes (e.g., different MAP kinase isoforms, splice variants, mutant genes etc.) with one siNA molecule. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific MAP kinase RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.
• In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplexes containing about 19 base pairs between oligonucleotides comprising about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24 or 25) nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplexes with overhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21-nucleotide duplexes with about 19 base pairs and 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs.
• In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for MAP kinase expressing nucleic acid molecules, such as RNA encoding a MAP kinase protein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.
• In one embodiment, a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, and/or bioavailability. For example, a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, a siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percent modification can be based upon the total number of nucleotides present in the single stranded siNA molecules. Likewise, if the siNA molecule is double stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long.
• In one embodiment, a siNA molecule of the invention does not contain any ribonucleotides. In another embodiment, a siNA molecule of the invention comprises one or more ribonucleotides.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of the MAP kinase gene or a portion thereof, and wherein the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the MAP kinase gene or a portion thereof.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and wherein each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of the MAP kinase gene or a portion thereof, and wherein the siNA further comprises a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the MAP kinase gene or a portion thereof.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the antisense region and the sense region each comprise about 19 to about 23 nucleotides, and wherein the antisense region comprises at least about 19 nucleotides that are complementary to nucleotides of the sense region.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule comprises a sense region and an antisense region and wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the MAP kinase gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. The sense region can be connected to the antisense region via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule comprises a sense region and an antisense region and wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the MAP kinase gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, wherein the siNA molecule has one or more modified pyrimidine and/or purine nucleotides. In one embodiment, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides, or 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In another embodiment of any of the above described siNA molecules, any nucleotides present in a non-complementary region of the sense strand (e.g. overhang region) are 2′-deoxy nucleotides.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule, and wherein the fragment comprising the sense region includes a terminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the fragment comprising the sense region. In one embodiment, the terminal cap moiety is an inverted deoxy abasic moiety or glyceryl moiety. In one embodiment, each of the two fragments of the siNA molecule comprises about 21 nucleotides.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule comprises a sense region and an antisense region and wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of RNA encoded by the MAP kinase gene and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the purine nucleotides present in the antisense region comprise 2′-deoxy-purine nucleotides. In another embodiment, the purine nucleotides present in the antisense region comprise 2′-O-methyl purine nucleotides. In either of the above embodiments, the antisense region comprises a phosphorothioate internucleotide linkage at the 3′ end of the antisense region. In an alternative embodiment, the antisense region comprises a glyceryl modification at the 3′ end of the antisense region. In another embodiment of any of the above described siNA molecules, any nucleotides present in a non-complementary region of the antisense strand (e.g. overhang region) are 2′-deoxy nucleotides.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein the siNA molecule is assembled from two separate oligonucleotide fragments each comprising 21 nucleotides, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule, and wherein about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule and wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine. In another embodiment, all 21 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the MAP kinase gene. In another embodiment, 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the MAP kinase gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a MAP kinase RNA sequence (e.g., wherein said target RNA sequence is encoded by a MAP kinase gene or a gene involved in the MAP kinase pathway), wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the double-stranded siNA molecule is about 21 nucleotides long.
• In one embodiment, the invention features a medicament comprising a siNA molecule of the invention.
• In one embodiment, the invention features an active ingredient comprising a siNA molecule of the invention.
• In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to down-regulate expression of a MAP kinase gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long.
• The invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of a MAP kinase RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, the nucleotide sequence of the antisense strand of the double-stranded siNA molecule is complementary to the nucleotide sequence of the MAP kinase RNA which encodes a protein or a portion thereof. In one embodiment, each strand of the siNA molecule comprises about 19 to about 29 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siNA molecule. In another embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. In one embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In one embodiment, wherein the sense strand comprises a 3′-end and a 5′-end, a terminal cap moiety (e.g., an inverted deoxy abasic moiety) is present at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand. In one embodiment, the antisense strand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methyl purine nucleotides. In one embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-O-methyl purine nucleotides. In one embodiment, the antisense strand comprises a phosphorothioate internucleotide linkage at the 3′ end of the antisense strand. In another embodiment, the antisense strand comprises a glyceryl modification at the 3′ end. In another embodiment, the 5′-end of the antisense strand optionally includes a phosphate group. In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of MAP kinase RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the 5′-untranslated region or a portion thereof of the MAP kinase RNA. In another embodiment, the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the MAP kinase RNA or a portion thereof.
• In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of MAP kinase RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein each of the two strands of the siNA molecule comprises 21 nucleotides. In one embodiment, about 19 nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule and at least two 3′ terminal nucleotides of each strand of the siNA molecule are not base-paired to the nucleotides of the other strand of the siNA molecule. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule are 2′-deoxy-pyrimidines, such as 2′-deoxy-thymidine. In another embodiment, each strand of the siNA molecule is base-paired to the complementary nucleotides of the other strand of the siNA molecule. In one embodiment, about 19 nucleotides of the antisense strand are base-paired to the nucleotide sequence of the MAP kinase RNA or a portion thereof. In another embodiment, 21 nucleotides of the antisense strand are base-paired to the nucleotide sequence of the MAP kinase RNA or a portion thereof.
• In one embodiment, the invention features a composition comprising a siNA molecule of the invention and a pharmaceutically acceptable carrier or diluent.
• In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a MAP kinase gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of MAP kinase RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.
• In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity in humans.
• In any of the embodiments of siNA molecules described herein, the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.
• One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to an RNA or DNA sequence encoding MAP kinase and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbone modified internucleotide linkage having Formula I:
• 
• wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y, and Z are optionally not all O.
• The chemically-modified internucleotide linkages having Formula I, for example, wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified internucleotide linkages having Formula I at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In another embodiment, a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-VII.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II:
• 
• wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.
• The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula II at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end of the sense strand, the antisense strand, or both strands.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III:
• 
• wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.
• The chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, the antisense strand, or both strands.
• In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a 5′-terminal phosphate group having Formula IV:
• 
• wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; wherein each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo; and wherein W, X, Y and Z are not all O.
• In one embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand, for example, a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3)nucleotide 3′-terminal nucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target-complementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I-VII.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In yet another embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.
• In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.
• In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.
• In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, being present in the same or different strand.
• In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages in each strand of the siNA molecule.
• In another embodiment, the invention features a siNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one or both siNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage.
• In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand is about 18 to about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in length, wherein the duplex has about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs. In another embodiment, a siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 base pairs and a 2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear 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 about 2 nucleotides.
• In another embodiment, a siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.
• In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, 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.
• In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having Formula V:
• 
• wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2.
• In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI:
• 
• wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siNA molecule of the invention.
• In another embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula VII:
• 
• wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having Formula I, and R1, R2 or R3 serves as points of attachment to the siNA molecule of the invention.
• In another embodiment, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0 and is the point of attachment to the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as “glyceryl” (forexample modification6 inFIG. 10).
• In another embodiment, a moiety having any of Formula V, VI or VII of the invention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of a siNA molecule of the invention. For example, a moiety having Formula V, VI or VII can be present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense strand, the sense strand, or both antisense and sense strands of the siNA molecule. In addition, a moiety having Formula VII can be present at the 3′-end or the 5′-end of a hairpin siNA molecule as described herein.
• In another embodiment, a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula VI or VI is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.
• In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.
• In another embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said antisense region are 2′-deoxy nucleotides.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where one or more purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and inverted deoxy abasic modifications that are optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the chemically-modified short interfering nucleic acid molecule comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown inFIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown inFIGS. 4 and 5 and Tables III and IV herein.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where one or more purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and inverted deoxy abasic modifications that are optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the chemically-modified short interfering nucleic acid molecule comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown inFIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown inFIGS. 4 and 5 and Tables III and IV herein.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where one or more purine nucleotides present in the sense region are purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides), and inverted deoxy abasic modifications that are optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the siNA comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown inFIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown inFIGS. 4 and 5 and Tables III and IV herein.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and for example where one or more purine nucleotides present in the sense region are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides), and wherein inverted deoxy abasic modifications are optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the chemically-modified short interfering nucleic acid molecule comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein one or more purine nucleotides present in the antisense region are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides), and a terminal cap modification, such as any modification described herein or shown inFIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages.
• In another embodiment, any modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methyl nucleotides.
• In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against a MAP kinase inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3′-end and 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al., U.S. Ser. No. 10/201,394, incorporated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example, in animal models as are generally known in the art.
• In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of ≧2 nucleotides in length, for example 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. (See, for example, Gold et al., 1995,Annu. Rev. Biochem.,64, 763; Brody and Gold, 2000,J. Biotechnol.,74, 5; Sunday, 2000,Curr. Opin. Mol. Ther.,2, 100; Kusser, 2000,J. Biotechnol.,74, 27; Hermann and Patel, 2000,Science,287, 820; and Jayasena, 1999,Clinical Chemistry,45, 1628.)
• In yet another embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser,Nucleic Acids Res.1990, 18:6353 andNucleic Acids Res.1987, 15:3113; Cload and Schepartz,J. Am. Chem. Soc.1991, 113:6324; Richardson and Schepartz,J. Am. Chem. Soc.1991, 113:5109; Ma et al.,Nucleic Acids Res.1993, 21:2585 andBiochemistry1993, 32:1751; Durand et al.,Nucleic Acids Res.1990, 18:6353; McCurdy et al.,Nucleosides&Nucleotides1991, 10:287; Jschke et al.,Tetrahedron Lett.1993, 34:301; Ono et al.,Biochemistry1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine,J. Am. Chem. Soc.1991, 113:4000, all hereby incorporated by reference herein. A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.
• In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides. For example, a siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA comprise separate oligonucleotides that do not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotides. In another example, a siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotide. Applicant has surprisingly found that the presence of ribonucleotides (e.g., nucleotides having a 2′-hydroxyl group) within the siNA molecule is not required or essential to support RNAi activity. As such, in one embodiment, all positions within the siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.
• In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a 2′,3′-cyclic phosphate). In another embodiment, the single stranded siNA molecule of the invention comprises about 19 to about 29 nucleotides. In yet another embodiment, the single stranded siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein. For example, all the positions within the siNA molecule can include chemically-modified nucleotides such as nucleotides having any of Formulae I-VII, or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.
• In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown inFIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.
• In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the siNA are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown inFIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.
• In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the siNA are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and a terminal cap modification, such as any modification described herein or shown inFIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.
• In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the siNA are locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides), and a terminal cap modification, such as any modification described herein or shown inFIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.
• In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the siNA are 2′-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-methoxyethyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown inFIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group.
• In another embodiment, any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.
• In one embodiment, the invention features a method for modulating the expression of a MAP kinase gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the MAP kinase gene in the cell.
• In one embodiment, the invention features a method for modulating the expression of a MAP kinase gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene and wherein the sense strand sequence of the siNA comprises a sequence identical to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the MAP kinase gene in the cell.
• In another embodiment, the invention features a method for modulating the expression of more than one MAP kinase gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the MAP kinase genes in the cell.
• In another embodiment, the invention features a method for modulating the expression of more than one MAP kinase gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene and wherein the sense strand sequence of the siNA comprises a sequence identical to the sequence of the target RNA; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the MAP kinase genes in the cell.
• In one embodiment, siNA molecules of the invention are 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 target cells from a patient are extracted. These extracted cells are contacted with 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. In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase gene in that organism.
• In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene and wherein the sense strand sequence of the siNA comprises a sequence identical to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase gene in that organism.
• In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase genes in that organism.
• In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in an organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the MAP kinase gene in the organism.
• In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the MAP kinase genes; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the MAP kinase genes in the organism.
• In one embodiment, the invention features a method for modulating the expression of a MAP kinase gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the MAP kinase gene in the cell.
• In another embodiment, the invention features a method for modulating the expression of more than one MAP kinase gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) contacting the siNA molecule with a cell in vitro or in vivo under conditions suitable to modulate the expression of the MAP kinase genes in the cell.
• In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) contacting the siNA molecule with a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase gene in that organism.
• In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the MAP kinase genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the MAP kinase genes in that organism.
• In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in an organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the MAP kinase gene in the organism.
• In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the MAP kinase gene; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the MAP kinase genes in the organism.
• In one embodiment, the invention features a method of modulating the expression of a MAP kinase gene in an organism comprising contacting the organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the MAP kinase gene in the organism.
• In another embodiment, the invention features a method of modulating the expression of more than one MAP kinase gene in an organism comprising contacting the organism with one or more siNA molecules of the invention under conditions suitable to modulate the expression of the MAP kinase genes in the organism.
• The siNA molecules of the invention can be designed to down-regulate or inhibit target (MAP kinase) gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).
• In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as MAP kinase family genes. As such, siNA molecules targeting multiple MAP kinase targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, the progression and/or maintenance of cancer.
• In one embodiment, siNA molecule(s) and/or methods of the invention are used to down-regulate the expression of gene(s) that encode RNA referred to by Genbank Accession, for example MAP kinase genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example, Genbank Accession Nos. shown in Table I.
• In one embodiment, the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In one embodiment, the siNA molecules of (a) have strands of a fixed length, for example, about 23 nucleotides in length. In another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.
• In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4N, where N represents the number of base paired nucleotides in each of the siNA construct strands (eg. for a siNA construct having 21 nucleotide sense and antisense strands with 19 base pairs, the complexity would be 419); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target MAP kinase RNA sequence. In another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 7 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of MAP kinase RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target MAP kinase RNA sequence. The target MAP kinase RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.
• In another embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In one embodiment, the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In another embodiment, the siNA molecules of (b) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.
• By “target site” is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.
• By “detectable level of cleavage” is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.
• In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for diagnosing a disease or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease or condition in the subject. In another embodiment, the invention features a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds. In yet another embodiment, the invention features a method for reducing or preventing tissue rejection in a subject comprising administering to the subject a composition of the invention under conditions suitable for the reduction or prevention of tissue rejection in the subject.
• In another embodiment, the invention features a method for validating a MAP kinase gene target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a MAP kinase target gene; (b) introducing the siNA molecule into a cell, tissue, or organism under conditions suitable for modulating expression of the MAP kinase target gene in the cell, tissue, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, or organism.
• In another embodiment, the invention features a method for validating a MAP kinase target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a MAP kinase target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the MAP kinase target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.
• By “biological system” is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human, animal, plant, insect, bacterial, viral or other sources, wherein the system comprises the components required for RNAi activity. The term “biological system” includes, for example, a cell, tissue, or organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.
• By “phenotypic change” is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA). Such detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.
• In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a MAP kinase target gene in a biological system, including, for example, in a cell, tissue, or organism. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one MAP kinase target gene in a biological system, including, for example, in a cell, tissue, or organism.
• In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified. In another embodiment, the cell containing a siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siNA molecule of the invention is a human cell.
• In one embodiment, the synthesis of a siNA molecule of the invention, which can be chemically-modified, comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.
• In one embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions using an alkylamine base such as methylamine. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example, using acidic conditions.
• In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.
• In another embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In one embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.
• In another embodiment, the invention features a method for making a double-stranded siNA molecule in a single synthetic process comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein aterminal 5′-protecting group, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.
• In another embodiment, the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein in their entirety.
• In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.
• In another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.
• In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the sense and antisense strands of the siNA construct.
• In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.
• In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell.
• In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target DNA sequence within a cell.
• In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.
• In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence.
• In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.
• In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.
• In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against a MAP kinase in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.
• In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against MAP kinase comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity.
• In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a MAP kinase target RNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target RNA.
• In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a MAP kinase target DNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target DNA.
• In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the cellular uptake of the siNA construct.
• In another embodiment, the invention features a method for generating siNA molecules against MAP kinase with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved cellular uptake.
• In one embodiment, the invention features siNA constructs that mediate RNAi against a MAP kinase, wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394 incorporated by reference herein.
• In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing a conjugate into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; polyamines, such as spermine or spermidine; and others.
• In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing an excipient formulation to a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, and others.
• In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing nucleotides having any of Formulae I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.
• In another embodiment, 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 2,000 to about 50,000 daltons (Da).
• The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include a siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, U.S. Pat. No. 6,395,713). The kit can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996). Such a kit can also include instructions to allow a user of the kit to practice the invention.
• The term “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” as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner; see for example Bass, 2001,Nature,411, 428-429; Elbashir et al., 2001,Nature,411, 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, 1833-1837; Jenuwein, 2002,Science,297, 2215-2218; 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 & Bartel, 2002,Science,297, 1831). Non limiting examples of siNA molecules of the invention are shown inFIGS. 4-6, and Tables II, III, and IV herein. For example the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs); the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having 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 nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having 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 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 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.,110, 563-574 and Schwarz et al., 2002,Molecular Cell,10, 537-568), or 5′,3′-diphosphate. In certain embodiment, the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides “siMON.” As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, 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, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure to alter gene expression (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).
• By “modulate” is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more 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 modulator. For example, the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
• By “inhibit”, “down-regulate”, or “reduce”, it is meant that 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, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction 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.
• By “gene” or “target gene” is meant, a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. 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.
• By “MAP kinase” is meant, any mitogen activated protein kinase (MAP kinase) polypeptide, protein and/or a polynucleotide encoding a MAP kinase protein (such as polynucleotides referred to by Genbank Accession number in Table I or any other MAP kinase transcript derived from a MAP kinase gene, e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38). As used herein, the term “MAP kinase gene” is meant to refer to any polynucleotide included in a group of MAP kinase genes, such as c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38).
• By “MAP kinase protein” is meant, any mitogen activated protein kinase (MAP kinase) peptide or protein or a component thereof, wherein the peptide or protein is encoded by a MAP kinase gene (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38).
• By “highly conserved sequence region” is meant a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.
• By “sense region” is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.
• By “antisense region” is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule.
• By “target nucleic acid” is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA.
• By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. 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., 1987,CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986,Proc. Nat. Acad. Sci. USA83:9373-9377; Turner et al., 1987,J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly 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.
• The siRNA molecules of the invention represent a novel therapeutic approach to treat a variety of pathologic indications or other conditions, including oncology and proliferation related indications and conditions such as multidrug resistant cancers, breast cancer, cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, cancers of the retina, cancers of the esophagus, multiple myeloma, ovarian cancer, melanoma, colorectal cancer, hepatocellular carcinoma, lung cancer, bladder cancer, pancreatic cancer, prostate cancer, glioblastoma; obesity and insulin resistance (e.g. type I and II diabetes); inflammatory disorders such as asthma, septic shock, rheumatoid arthritis, psoriasis, inflammatory bowl syndrome and any other diseases or conditions that are related to or will respond to the levels of MAP kinase in a cell or tissue, alone or in combination with other therapies. The reduction of MAP kinase expression (specifically MAP kinase gene RNA levels) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.
• In one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently about 18 to about 24 nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 17 to about 23 base pairs (e.g., about 17, 18, 19, 20, 21, 22 or 23). In yet another embodiment, siNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., 38, 39, 40, 41, 42, 43 or 44) nucleotides in length and comprising about 16 to about 22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs. Exemplary siNA molecules of the invention are shown in Table II. Exemplary synthetic siNA molecules of the invention are shown in Tables III and IV and/orFIGS. 4-5.
• As used herein “cell” 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. 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.
• The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables II-III and/orFIGS. 4-5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures. Furthermore, the chemically modified constructs described in Table IV can be applied to any siNA sequence of the invention.
• In another aspect, the invention provides mammalian cells containing one or more siNA molecules of this invention. The one or more siNA molecules can independently be targeted to the same or different sites.
• By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety. The terms 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.
• By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to 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 “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.
• The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little 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).
• The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.
• The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein (e.g., cancers and other proliferative conditions). For example, to treat a particular disease or condition, the siNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
• In a further embodiment, the siNA molecules can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules could be used in combination with one or more known therapeutic agents to treat a disease or condition. Non-limiting examples of other therapeutic agents that can be readily combined with a siNA molecule of the invention are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.
• In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors are described in 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.
• In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
• In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in Table I.
• In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.
• In another aspect of the invention, siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units 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. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular 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.
• By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
• Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands,strand1 andstrand2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.
• FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.
• FIG. 3 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 which 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.
• FIG. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siNA constructs.
• FIG. 4A: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand.
• FIG. 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the sense and antisense strand.
• FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand.
• FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand.
• FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand.
• FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s” connects the (N N) nucleotides in the antisense strand. The antisense strand of constructs A-F comprise sequence complementary to any target nucleic acid sequence of the invention. Furthermore, when a glyceryl moiety (L) is present at the 3′-end of the antisense strand for any construct shown inFIG. 4 A-F, the modified internucleotide linkage is optional.
• FIG. 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described inFIG. 4A-F to a c-JUN siNA sequence. Such chemical modifications can be applied to any sequence herein, such as any MAP kinase sequence.
• FIG. 6 shows non-limiting examples of different siNA constructs of the invention. The examples shown (constructs1,2, and3) 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.Constructs1 and2 can be used independently for RNAi activity.Construct2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown inconstruct2 can comprise a biodegradable linker that results in the formation ofconstruct1 in vivo and/or in vitro. In another example, construct3 can be used to generateconstruct2 under the same principle wherein a linker is used to generate the active siNA construct2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct1 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 in vitro.
• FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.
• FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined MAP kinase target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.
• FIG. 7B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for a MAP kinase target sequence and having self-complementary sense and antisense regions.
• FIG. 7C: The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-terminal nucleotide overhang results from the transcription, for example by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology,29, 505-508.
• FIG. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.
• FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined MAP kinase target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).
• FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.
• FIG. 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.
• FIG. 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.
• FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.
• FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted into vectors such that (FIG. 9C) transfection of a vector into cells results in the expression of the siNA.
• FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.
• FIG. 9E: The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.
• FIG. 10 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 3′-end 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′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I-VII or any combination thereof.
• FIG. 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistance 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 in 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). 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.
• FIG. 12 shows a non-limiting example of reduction of p38 mRNA in A549 cells mediated by siNAs that target p38 mRNA. A549 cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. A screen of siNA constructs comprising ribonucleotides and 3′-terminal dithymidine caps was compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown in the figure, the siNA constructs significantly reduce p38 RNA expression.
• FIG. 13 shows a non-limiting example of reduction of JNK1 mRNA in A549 cells mediated by siNAs that target JNK1 mRNA. A549 cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. A screen of siNA constructs comprising ribonucleotides and 3′-terminal dithymidine caps was compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown in the figure, the siNA constructs significantly reduce JNK1 RNA expression.
DETAILED DESCRIPTION OF THE INVENTIONMechanism of Action of Nucleic Acid Molecules of the Invention
• The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limited to siRNA only and can be applied to siNA as a whole. By “improved capacity to mediate RNAi” or “improved RNAi activity” is meant to include 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 RNA siRNA or a 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.
• RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998,Nature,391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999,Trends Genet.,15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
• The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001,Nature,409, 363). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001,Science,293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA 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 though cellular mechanisms 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). As such, siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.
• RNAi has been studied in a variety of systems. Fire et al., 1998,Nature,391, 806, were the first to observe RNAi inC. elegans. Wianny and Goetz, 1999,Nature Cell Biol.,2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,Nature,404, 293, describe RNAi inDrosophilacells transfected with dsRNA. Elbashir et al., 2001,Nature,411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work inDrosophilaembryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3′-terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001,EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell,107, 309); however, siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.
Synthesis of Nucleic Acid Molecules
• Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be 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 Enzymology211, 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. 6,001,311. All of these references are incorporated herein by reference. 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. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 minute coupling step for 2′-O-methylated nucleotides and a 45 second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.
• Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
• The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc.,109, 7845; Scaringe et al., 1990,Nucleic Acids Res.,18, 5433; and Wincott et al., 1995,Nucleic Acids Res.23, 2677-2684 Wincott et al., 1997,Methods Mol. Bio.,74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 minute coupling step for alkylsilyl protected nucleotides and a 2.5 minute coupling step for 2′-O-methylated nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.
• Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 hours, the oligomer is quenched with 1.5 M NH₄HCO₃.
• Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 minutes. The vial is brought to room temperature. TEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 minutes. The sample is cooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.
• For purification of the trityl-on oligomers, the quenched NH₄.HCO₃solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
• The average stepwise coupling yields are typically >98% (Wincott et al., 1995Nucleic Acids Res.23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.
• Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992,Science256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991,Nucleic Acids Research19, 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.
• The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.
• A siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.
• The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992,TIBS17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser.31, 163). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.
• In another aspect of the invention, siNA molecules of the invention are expressed from transcription units 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. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.
Optimizing Activity of the Nucleic Acid Molecule of the Invention.
• Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990Nature344, 565; Pieken et al., 1991,Science253, 314; Usman and Cedergren, 1992,Trends in Biochem. Sci.17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.
• There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992,TIBS.17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser.31, 163; Burgin et al., 1996,Biochemistry,35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al.Nature,1990, 344, 565-568; Pieken et al.Science,1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci.,1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem.,270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998,Tetrahedron Lett.,39, 1131; Earnshaw and Gait, 1998,Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998,Annu. Rev. Biochem.,67, 99-134; and Burlina et al., 1997,Bioorg. Med. Chem.,5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.
• While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.
• Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995,Nucleic Acids Res.23, 2677; Caruthers et al., 1992,Methods in Enzymology211, 3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.
• In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998,J. Am. Chem. Soc.,120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).
• In another embodiment, 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 pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example, proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.
• The term “biodegradable linker” as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a 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′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-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.
• The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
• The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
• The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.
• Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
• In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.
• Use of the nucleic acid-based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.
• In another aspect a siNA molecule of the invention comprises one or more 5′ and/or a 3′-cap structure, for example on only the sense siNA strand, the antisense siNA strand, or both siNA strands.
• By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). 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. Non-limiting examples of the 5′-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-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; 1,4-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, glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-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; 1,4-butanediol phosphate; 5′-amino; bridging and/ornon-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993,Tetrahedron49, 1925; incorporated by reference herein).
• By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. 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 base at the 1′-position.
• An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl 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, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino or SH.
• Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group 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 include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR, where R is either alkyl, aryl, alkylaryl or hydrogen.
• By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994,Nucleic Acids Res.22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996,Biochemistry,35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.
• In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, inModern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994,Novel Backbone Replacements for Oligonucleotides, inCarbohydrate Modifications in Antisense Research, ACS, 24-39.
• By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, see for example Adamic et al., U.S. Pat. No. 5,998,203.
• By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1′ carbon of β-D-ribo-furanose.
• By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.
• In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH₂or 2′-O—NH₂, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.
• Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
Administration of Nucleic Acid Molecules
• A siNA molecule of the invention can be adapted for use to treat, for example, oncology and proliferation related indications and conditions such as multidrug resistant cancers, breast cancer, cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, cancers of the retina, cancers of the esophagus, multiple myeloma, ovarian cancer, melanoma, colorectal cancer, hepatocellular carcinoma, lung cancer, bladder cancer, pancreatic cancer, prostate cancer, glioblastoma; obesity and insulin resistance (e.g. type I and II diabetes); inflammatory disorders such as asthma, septic shock, rheumatoid arthritis, psoriasis, inflammatory bowl syndrome and any other diseases or conditions that are related to or will respond to the levels of MAP kinase in a cell or tissue, alone or in combination with other therapies. For example, a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992,Trends Cell Bio.,2, 139;Delivery 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, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery 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), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 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). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., 1999,Clin. Cancer Res.,5, 2330-2337 and Barry et al., International PCT Publication No. WO 99/31262. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.
• Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art.
• The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
• A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic 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, 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, pharmacological 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.
• By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cells producing excess MAP kinase.
• By “pharmaceutically acceptable formulation” is meant a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999,Fundam. Clin. Pharmacol.,13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D F et al, 1999,Cell Transplant,8, 47-58) (Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry,23, 941-949, 1999). 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., 1999,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.
• The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al.Chem. Rev.1995, 95, 2601-2627; Ishiwata et al.,Chem. Pharm. Bull.1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al.,Science1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta,1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al.,J. Biol. Chem.1995, 42, 24864-24870; Choi et al., 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). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
• The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.
• A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
• The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
• 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 monostearate or glyceryl distearate can be employed.
• 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.
• 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.
• 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 anti-oxidant such as ascorbic acid
• Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.
• 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, naturally-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.
• Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations 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.
• 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.
• 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.
• Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
• It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
• For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
• The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
• In one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987,J. Biol. Chem.262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). In another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980,Cell,22, 611-620; Connolly et al., 1982,J. Biol. Chem.,257, 939-945). Lee and Lee, 1987,Glycoconjugate J.,4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981,J. Med. Chem.,24, 1388-1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser. No. 60/362,016, filed Mar. 6, 2002.
• Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985,Science,229, 345; McGarry and Lindquist, 1986,Proc. Natl. Acad. Sci., USA83, 399; Scanlon et al., 1991,Proc. Natl. Acad. Sci. USA,88, 10591-5; Kashani-Sabet et 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., 1990Science,247, 1222-1225; Thompson et al., 1995,Nucleic Acids Res.,23, 2259; Good et al., 1997,Gene Therapy,4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by an enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser.,27, 15-6; Taira et al., 1991,Nucleic Acids Res.,19, 5125-30; Ventura et al., 1993,Nucleic Acids Res.,21, 3249-55; Chowrira et al., 1994,J. Biol. Chem.,269, 25856.
• In another aspect of the invention, RNA molecules of the present invention can be expressed 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. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular 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).
• In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant invention. The expression vector can encode one or both strands of a siNA duplex, or a single self-complementary strand that self hybridizes into a 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).
• In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siNA of the invention and/or an intron (intervening sequences).
• 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). 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. USA,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. USA,89, 10802-6; Chen et al., 1992,Nucleic Acids Res.,20, 4581-9; Yu et al., 1993,Proc. Natl. Acad. Sci. USA,90, 6340-4; L'Huillier et al., 1992,EMBO J.,11, 4411-8; Lisziewicz et al., 1993,Proc. Natl. Acad. Sci. U.S. 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).
• In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.
• In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.
• In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.
MAP Kinase Biology and Biochemistry
• The mitogen-activated protein kinases (MAPKs) have been at the forefront of a rapid advance in the understanding of cellular events in growth factor and cytokine receptor signaling. The MAP kinases (also referred to as extracellular signal-regulated protein kinases, or ERKs) are the terminal enzymes in a three-kinase cascade. The reiteration of three-kinase cascades for related but distinct signaling pathways gave rise to the concept of a MAPK pathway as a modular, multifunctional signaling element that acts sequentially within one pathway, where each enzyme phosphorylates and thereby activates the next member in the sequence. A typical MAPK pathway thus consists of three protein kinases: a MAPK kinase kinase (or MEKK) that activates a MAPK kinase (or MEK) which, in turn, activates a MAPK/ERK enzyme. Each of the MAPK/ERK, JNK and p38 cascades consists of a three-enzyme module that includes MEKK, MEK and an ERK or MAPK superfamily member. A variety of extracellular signals triggers initial events upon association with their respective cell surface receptors and this signal is then transmitted to the interior of the cell where it activates the appropriate cascades (see for exampleFIG. 12).
• The identification of distinct MAPK cascades that are conserved across all eukaryotes indicates that the MAPK module has been adapted for interpretation of a diverse array of extracellular signals. Although mitogen activation of the MAPK subfamily (e.g., ERK1 and ERK2) has dominated efforts to understand MAPK signaling, increasing appreciation of the role of the stress-activated kinases, JNK and p38, illustrates the diverse nature of the MAPK superfamily of enzymes. Although sequence similarities among components of the individual MAPK modules used for activation of ERK1/2, JNKs and p38 are considerable, the fidelity that is maintained in order to translate specific extracellular signals into discrete physiological responses illustrates the selective adaptation of each MAPK pathway. The MAPK superfamily of enzymes is a critical component cellular regulative processes that coordinates incoming signals generated by a variety of extracellular and intracellular mediators. Specific phosphorylation and activation of enzymes in the MAPK pathway transmits the signal down the cascade, resulting in phosphorylation of many proteins with substantial regulatory functions throughout the cell, including other protein kinases, transcription factors, cytoskeletal proteins and other enzymes. The diversity of signals that culminates in MAPK activation indicates that these enzymes are not dedicated to regulation of any single growth factor, hormone or cytokine system. Instead, MAPKs—like cAMP-dependent protein kinase (PKA) and Ca²⁺- and phospholipid-dependent protein kinases (PKC) serve many signaling purposes. Because activation of the MAPK pathways are triggered to varying extents by a large number of receptor systems, temporal and spatial differences are critical to determining ligand- and cell-type-specific functions.
• Following activation of cells with an appropriate extracellular stimuli, the signal is transmitted to the canonical MAPK module comprising three protein kinases. The progression of events for each enzyme cascade is the same, although specific isoforms of each enzyme appear to confer the required specificity within each pathway. The first enzyme in the module is a MEKK enzyme, of which Raf and its isoforms are one example. The MEKK enzymes comprise Ser/Thr protein kinases that activate the MEK enzymes by phosphorylating two serine or threonine residues within a Ser-X-X-X-Ser/Thr motif. Once activated, the MEK enzymes, which are hybrid function Ser/Thr/Tyr protein kinases, phosphorylate the MAPK/ERK enzymes on Thr and Tyr residues within the Thr-X-Tyr (TXY) consensus sequence. A critical and common feature of the MAPK superfamily of enzymes is that they are activated upon dual phosphorylation within a TXY consensus sequence present in the activation loop of the catalytic domain. The central amino acid differs for each MAPK superfamily member, corresponding to Glu for ERK1/2, Gly for p38/HOG and Pro for JNK/SAPK, although MEK specificity is not limited to these particular residues. Phosphorylation at only one of the two positions does not appear to activate the enzyme, although it may prime the kinase domain for receipt of the second phosphorylation event.
• ERK1 and ERK2 were the first members of the MAPK superfamily whose cDNAs were cloned and the signaling cascades that lead to their activation characterized. Potent activation of ERK1 and ERK2 can be initiated through activation of transmembrane receptors with intrinsic protein tyrosine kinase (PTK) activity. Binding of extracellular ligands to their respective cell surface receptors results in receptor autophosphorylation and enhanced PTK activity. The subsequent association of the Src homology 2 (SH2) domains of adaptor proteins such as Grb2 and Shc with the autophosphorylated receptors, or with additional docking proteins, provides the molecular interactions that bring the required signal transduction molecules into close proximity with each other. Receptors without intrinsic PTK activity but which comprise sites for tyrosine phosphorylation can also activate the cascade via association of their phosphotyrosine residues with adaptor molecules. For example, the SH3 domain of Grb2 binds a proline-rich region of the guanine nucleotide-exchange protein SOS which, in turn, increases the association of Ras with GTP. The GTP-bound form of Ras binds to Raf (a MAPK kinase) isoforms, including C-Raf-1, B-Raf and A-Raf. This action targets Raf to the membrane, where its protein kinase activity is increased by phosphorylation. MAPK kinases (MEK1 and MEK2), are phosphorylated and activated by Raf. MEK1 and MEK2 are dual-specificity protein kinases that dually phosphorylate the ERK enzymes (corresponding to Thr¹⁸³and Tyr¹⁸⁵of p42ERK2), thereby increasing their enzymatic activity by approximately 1,000-fold over the activity found with the basal or monophosphorylated forms. Phosphorylation of these residues causes closure of the kinase active site and induces conformational changes necessary for high activity.
• MAPK mutants, lacking either a lysine required for catalytic activity or the prerequisite TXY phosphorylation sites, can inhibit signaling by the native enzymes in cells. In the case of ERK1 and ERK2, these mutants have been used with repeated success. For example, mutant ERK2 completely blocks proliferation in response to epidermal growth factor (EGF) and v-Raf, and partially blocks induction by serum or small t antigen. ERK1 antisense mRNA and an ERK1 phosphorylation site mutants interfere with thrombin-induced transcription as well as serum-dependent proliferation. These findings suggest an essential role in proliferation and transformation for the ERK/MAPK pathway.
• The JNK/SAPK and p38/HOG pathways are activated by ultraviolet light, cytokines, osmotic shock, inhibitors of DNA, RNA, and protein synthesis, and to a lesser extent by certain growth factors. This spectrum of regulators suggests that the enzymes are transducers of a variety of cellular stress responses. In contrast to activation of ERK1 and ERK2, upstream signal transduction mechanisms for the JNK and p38 cascades are less well understood. When transfected into mammalian cells, a diverse group of protein kinases including the mixed lineage kinases (MLKs) and relatives of the yeast Step 20p, such as the p21-activated kinases (PAKs) and germinal center kinase (GCK), cause activation of JNK/SAPK. Similarly, GTP-bound forms of the small GTP-binding proteins, Rac and Cdc42, activate the JNK/SAPK pathway and, to a lesser extent, the p38 pathway. Direct activation of both pathways by PAKs also has been demonstrated, suggesting that PAKs can be the relevant effectors for these small G proteins. The PAKs are homologs of the yeast kinases Step 20p and Shk1, enzymes upstream of the MAPK modules in yeast pheromone response pathways. Both yeast and mammalian protein kinases contain a binding site for Rac/Cdc42 and share the property of being activated in vitro through association with these small G proteins when in their GTP-bound states. In yeast, Step 20p is thought to phosphorylate and activate the MEKK isoform Ste11p, suggesting that MEKKs may be PAK targets. This summary of MAP kinase pathways has been adapted from Cobb and Schaefer, 1996, Promega Notes Magazine Number 59, page 37.
• The regulation of c-Jun transcriptional activity by Jun N-terminal kinase (JNK), ERK1, ERK2, and p38 kinases has become a paradigm for the understanding of how mitogen-activated protein (MAP) kinase signaling pathways elicit specific changes in gene transcription through selective phosphorylation of nuclear transcription factors. Selective phosphorylation of c-Jun by JNK is detected by a specific docking motif in c-Jun, the delta region, which enables JNK to physically interact with c-Jun. Analogous MAP kinase docking motifs have subsequently been found in several other transcription factors, indicating that this is a general mechanism for ensuring the specificity of signal transduction. Furthermore, genetic and biochemical studies in mice, flies and cultured cells have provided evidence that signals relayed by JNK through c-Jun regulate a wide range of cellular processes including cell proliferation, tumorigenesis, apoptosis and embryonic development. Despite these advances, in most cases, the genes or programs of gene expression downstream of JNK and c-Jun, which control these processes, have yet to be defined. One important process that is associated with JNK gene expression is the development of insulin resistance in obese subjects.
• Obesity is closely associated with insulin resistance and establishes the leading risk factor fortype 2 diabetes mellitus in mammals. The c-Jun amino-terminal kinases (JNKs) can interfere with insulin activity in cultured cells and are activated by inflammatory cytokines and free fatty acids molecules that have been implicated in the development oftype 2 diabetes. Hirosumi et al, 2002,Nature,420, 333-336, demonstrate that JNK activity is abnormally elevated in obesity. Furthermore, Hirosumi et al, supra have shown that an absence of JNK1 results in decreased adiposity with significantly improved insulin sensitivity and enhanced insulin receptor capacity in two different models of mouse obesity. Thus, JNK is a crucial mediator of obesity and insulin resistance and as such, provides a potential target for nucleic acid based therapeutics that modulate JNK gene expression.
• The transcription factor and oncogene, c-JUN, is implicated in several critical cell processes including cell proliferation, cell survival, and oncogenic transformation. Although it is broadly expressed in a wide variety of cell types, it plays an especially important role in hepatocytes. However, the precise role played by c-JUN in hepatocytes seems to depend on the differentiation state of this cell type. Adult differentiated hepatocytes depend on c-JUN for progression through the cell cycle. Deletion of c-JUN reduces the proliferation capacity of hepatocytes following partial hepatectomy. c-JUN is thought to be major component in the development of human hepatocellular carcinoma (HCC). HCC is the most common form of primary liver cancer. Chronic HCV infection is a major risk factor for HCC.
• The role of c-JUN in liver cancer has recently been investigated (Eferl et al., 2003,Cell,112, 181). These investigators deleted c-JUN and then induced liver cancer by chemical carcinogenesis. They observed that deletion of c-JUN dramatically interfered with liver tumor formation. Animal survival was markedly worse in c-JUN wild-type animals relative to deletion mutants. In particular, the number of apoptotic cells increased about five fold in tumors in the c-JUN deletion strain relative to the wild-type animals. Importantly, levels of the pro-apoptotic gene products such as p53 and noxa were elevated in the c-JUN deletion strain. c-JUN is likely to antagonize other pro-apoptotic genes such as TNF-a. Thus, by blocking p53 and its large family of dependent genes, c-JUN seems to promote tumor formation. Since a large fraction of chronically infected HCV patients develop hepatocellular carcinoma, c-JUN provides an attractive target for treating HCV infected patients to prevent or ameliorate hepatocellular carcinoma.
• Based upon the current understanding of MAP kinase pathways, the modulation of MAP kinase pathways is instrumental in the development of new therapeutics in, for example, the fields of inflammation, oncology, and metabolism. As such, modulation of a specific MAP kinase pathway using small interfering nucleic acid (siNA) mediated RNAi represents a novel approach to the treatment and study of diseases and conditions related to a specific MAP kinase activity and/or gene expression.
EXAMPLES
• The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.
Example 1Tandem Synthesis of siNA Constructs
• Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.
• After completing a tandem synthesis of a siNA oligo and its complement in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises aterminal 5′-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C18 cartridge.
• Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (seeFIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5′-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.
• Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example using a Waters C18 SepPak Ig cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H₂O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H₂O or 50 mM NaOAc. Failure sequences are eluted with 1CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H₂O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H₂O followed by 1 CV 1M NaCl and additional H₂O. The siNA duplex product is then eluted, for example, using 1CV 20% aqueous CAN.
• FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract only shows a single peak. Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.
Example 2Identification of Potential siNA Target Sites in Any RNA Sequence
• The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA construct to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.
Example 3Selection of siNA Molecule Target Sites in a RNA
• The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.
• 1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.
• 2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.
• 3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As incase 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.
• 4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.
• 5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.
• 6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.
• 7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end of the sequence (to yield 3′ UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.
• 8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Tables II and III). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.
• 9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.
• In an alternate approach, a pool of siNA constructs specific to a MAP kinase target sequence is used to screen for target sites in cells expressing MAP kinase (e.g., c-JUN) RNA, such as human kidney fibroblast (e.g., 293 cells), HeLa, or HepG2 cells. The general strategy used in this approach is shown inFIG. 9. A non-limiting example of such as pool is a pool comprising sequences having sense sequences comprising SEQ ID NOs. 1247-1427 and antisense sequences comprising SEQ ID NOs. 1428-1608 respectively. 293, HeLa, or HepG2 cells expressing MAP kinase (e.g., c-JUN) are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with MAP kinase (e.g., c-JUN) inhibition are sorted. The pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for exampleFIG. 7 andFIG. 8). The siNA from cells demonstrating a positive phenotypic change (e.g., decreased proliferation, decreased MAP kinase (e.g., c-JUN) mRNA levels or decreased MAP kinase (e.g., c-JUN) protein expression), are sequenced to determine the most suitable target site(s) within the target MAP kinase (e.g., c-JUN) RNA sequence.
Example 4MAP Kinase Targeted siNA Design
• siNA target sites were chosen by analyzing sequences of the MAP kinase (e.g., c-JUN) RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein. siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.
• Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constructs that possess both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for exampleFIG. 11).
Example 5Chemical Synthesis and Purification of siNA
• siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence. Generally, siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein in their entirety).
• In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl, 3′-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be used in conjunction with acid-labile 2′-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2′ chemistries can require different protecting groups, for example 2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference herein in its entirety).
• During solid phase synthesis, each nucleotide is added sequentially (3′- to 5′-direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support (e.g., controlled pore glass or polystyrene) using various linkers. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5′-end of the first nucleoside. The support is then washed and any unreacted 5′-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage. At the end of the nucleotide addition cycle, the 5′-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.
• Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in Deprotection and purification of the siNA can be performed as is generally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs. For example, applicant has observed that oligonucleotides comprising 2′-deoxy-2′-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35° C. for 30 minutes. If the 2′-deoxy-2′-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35° C. for 30 minutes, TEA-HF is added and the reaction maintained at about 65° C. for an additional 15 minutes.
Example 6RNAi In Vitro Assay to Assess siNA Activity
• An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting MAP kinase (e.g., c-JUN) RNA targets. The assay comprises the system described by Tuschl et al., 1999,Genes and Development,13, 3191-3197 and Zamore et al., 2000,Cell,101, 25-33 adapted for use with MAP kinase target RNA. ADrosophilaextract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate MAP kinase expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. TheDrosophilalysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 μM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.
• Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-³²P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by Phosphor Inager® quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.
• In one embodiment, this assay is used to determine target sites the MAP kinase RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the MAP kinase RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.
Example 7Nucleic Acid Inhibition of MAP Kinase Target RNA In Vivo
• siNA molecules targeted to the human MAP kinase (e.g., c-JUN) RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the MAP kinase (e.g., c-JUN) RNA are given in Table II and III.
• Two formats are used to test the efficacy of siNAs targeting MAP kinase (e.g., c-JUN). First, the reagents are tested in cell culture, for example using cultured human kidney fibroblast cells (e.g., 293, HeLa, or HepG2 cells), to determine the extent of RNA and protein inhibition. siNA reagents (e.g.; see Tables II and III) are selected against the MAP kinase (e.g., c-JUN) target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, 293, HeLa, or HepG2 cells. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 Taqman®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead siNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.
• Delivery of siNA to Cells
• Cells (e.g., 293, HeLa, or HepG2 cells) are seeded, for example, at 1×10⁵cells per well of a six-well dish in EGM-2 (BioWhittaker) the day before transfection. siNA (final concentration, for example 20 nM) and cationic lipid (e.g.,final concentration 2 μg/ml) are complexed in EGM basal media (BioWhittaker) at 37° C. for 30 mins in polystyrene tubes. Following vortexing, the complexed siNA is added to each well and incubated for the times indicated. For initial optimization experiments, cells are seeded, for example, at 1×10³in 96 well plates and siNA complex added as described. Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid. Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope.
• Taqman and Lightcycler Quantification of mRNA
• Total RNA is prepared from cells following siNA delivery, for example using Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays. For Taqman analysis, dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1×TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl₂, 300 mM each dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AmpliTaq Gold (PE-Applied Biosystems) and 10U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 min at 48° C., 10 min at 95° C., followed by 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/r×n) and normalizing to β-actin or GAPDH mRNA in parallel TaqMan reactions. For each gene of interest an upper and lower primer and a fluorescently labeled probe are designed. Real time incorporation of SYBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler. A standard curve is generated for each primer pair using control c RNA allularnd values are represented as relative expression to GAPDH in each sample.
Western Blotting
• Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991,Nucleic Acids Research,19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4° C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).
Example 8Models Useful to Evaluate the Down-Regulation of MAP Kinase Gene (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38) expressionCell Culture
• There are numerous cell culture systems that can be used to analyze reduction of MAP kinase levels either directly or indirectly by measuring downstream effects. For example, cultured human kidney fibroblast cells (e.g., 293 cells), HeLa, or HepG2 cells can be used in cell culture experiments to assess the efficacy of nucleic acid molecules of the invention. As such, cells treated with nucleic acid molecules of the invention (e.g., siNA) targeting MAP kinase RNA would be expected to have decreased MAP kinase expression capacity compared to matched control nucleic acid molecules having a scrambled or inactive sequence. In a non-limiting example, 293, HeLa, or HepG2 cells are cultured and MAP kinase expression is quantified, for example by time-resolved immuno fluorometric assay. MAP kinase messenger-RNA expression is quantitated with RT-PCR in cultured cells. Untreated cells are compared to cells treated with siNA molecules transfected with a suitable reagent, for example a cationic lipid such as lipofectamine, and MAP kinase protein and RNA levels are quantitated. Dose response assays are then performed to establish dose dependent inhibition of MAP kinase expression. In another non-limiting example, cell culture experiments are carried out as described by Aguirre et al., 2000,J. Biol. Chem.,275, 9047-9054.
• In several cell culture systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, et al., 1992,Mol. Pharmacology,41, 1023-1033). In one embodiment, siNA molecules of the invention are complexed with cationic lipids for cell culture experiments. siNA and cationic lipid mixtures are prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives are warmed to room temperature (about 20-25° C.) and cationic lipid is added to the final desired concentration and the solution is vortexed briefly. siNA molecules are added to the final desired concentration and the solution is again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex is serially diluted into DMEM following the 10 minute incubation.
Animal Models
• Evaluating the efficacy of anti-MAP kinase agents in animal models is an important prerequisite to human clinical trials. Obesity andtype 2 diabetes are the most prevalent and serious metabolic diseases in that they affect more than 50% of adults in the USA. These conditions are associated with a chronic inflammatory response characterized by abnormal inflammatory cytokine production, increased acute-phase reactants and other stress-induced molecules. Many of these alterations seem to be initiated and to reside within adipose tissue. Elevated production of tumor necrosis factor (TNF)-alpha by adipose tissue decreases sensitivity to insulin and has been detected in several experimental obesity models and obese humans. Free fatty acids (FFAs) are also implicated in the etiology of obesity-induced insulin resistance and diabetes. Because both TNF-alpha and FFAs are potent MAP kinase activators, Hirosumi et al., 2002,Nature,420, 333-336 determined whether obesity is associated with alterations in stress-activated and inflammatory responses through this pathway and whether MAP kinases are causally linked to aberrant metabolic control in this state. In this study, Hirosumi et al., describe dietary and genetic (ob/ob) mouse models of obesity useful in evaluating MAP kinase gene expression. Such transgenic mice are useful as models for obesity and insulin resistance and can be used to identify nucleic acid molecules of the invention that modulate MAP kinase gene (e.g., ERK1, ERK2, JNK1, JNK2, and/or p38) expression and gene function toward therapeutic use in treating obesity and insulin resistance (e.g. type I and II diabetes).
• The role of c-JUN in liver cancer has recently been investigated (Eferl et al., 2003,Cell,112, 181). These investigators deleted c-JUN and then induced liver cancer by chemical carcinogenesis. They observed that deletion of c-JUN dramatically interfered with liver tumor formation. Animal survival was markedly worse in c-JUN wild-type animals relative to deletion mutants. In particular, the number of apoptotic cells increased about five fold in tumors in the c-JUN deletion strain relative to the wild-type animals. Importantly, levels of the pro-apoptotic gene products such as p53 and noxa were elevated in the c-JUN deletion strain. c-JUN is likely to antagonize other pro-apoptotic genes such as TNF-a. Thus, by blocking p53 and its large family of dependent genes, c-JUN seems to promote tumor formation. Since a large fraction of chronically infected HCV patients develop hepatocellular carcinoma, c-JUN provides an attractive target for treating HCV infected patients to prevent or ameliorate hepatocellular carcinoma. The animal model described by Eferl et al., supra, can be used to evaluate siNA molecules of the invention for efficacy in inhibiting c-JUN expression in liver toward therapeutic use in preventing and/or treating hepatocellular carcinoma in human subjects.
• Because mitogen activated protein kinases (MAP kinases) are constituents of numerous signal transduction pathways, and are activated by protein kinase cascades, intense efforts are under way to develop and evaluate compounds that target components of MAPK pathways. Several of these inhibitors are effective in animal models of disease and have advanced to clinical trials for the treatment of inflammatory diseases, metabolic diseases, autoimmune diseases and cancer. The clinical utility of specifically targeting MAP kinase genes (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38) can be studied in animal models and clinical studies of inflammatory diseases, metabolic diseases, autoimmune diseases and cancer (see for example English et al., 2002,Trends in Pharmacological Sciences,23, 40-45).
Example 9RNAi Mediated Inhibition of p38 RNA Expression
• siNA constructs are tested for efficacy in reducing p38 RNA expression in, for example in A549 cells. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 hours in the continued presence of the siNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs was determined.
• In a non-limiting example, siNA constructs were screened for activity (seeFIG. 12) and compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown inFIG. 12, the siNA constructs significantly reduce p38 RNA expression. Leads generated from such a screen are then further assayed. In a non-limiting example, siNA constructs comprising chemical modifications described herein (e.g., having modifications comprising Formulae I-VII and/or those modifications described in Table IV are assayed for activity. These siNA constructs are compared to appropriate matched chemistry inverted controls. In addition, the siNA constructs are also compared to untreated cells, cells transfected with lipid and scrambled siNA constructs, and cells transfected with lipid alone (transfection control).
Example 10RNAi Mediated Inhibition of p38 RNA Expression
• siNA constructs are tested for efficacy in reducing JNK1 RNA expression in, for example in A549 cells. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 hours in the continued presence of the siNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs was determined.
• In a non-limiting example, siNA constructs were screened for activity (seeFIG. 13) and compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown inFIG. 13, the siNA constructs significantly reduce p38 RNA expression. Leads generated from such a screen are then further assayed. In a non-limiting example, siNA constructs comprise chemical modifications described herein (e.g., having modifications comprising Formulae I-VII and/or those modifications described in Table IV are assayed for activity). These siNA constructs are compared to appropriate matched chemistry inverted controls. In addition, the siNA constructs are also compared to untreated cells, cells transfected with lipid and scrambled siNA constructs, and cells transfected with lipid alone (transfection control).
Example 11Indications
• The present body of knowledge in MAP kinase research indicates the need for methods and compounds that can regulate MAP kinase gene (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38) product expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used to treat obesity and insulin resistance (e.g. type I and II diabetes), oncology and proliferation related indications and conditions, including cancers of the lung, bladder, colon, breast, prostate, retina, larynx, esophagus, liver (e.g., hepatocellular carcinoma), and ovary, along with lymphomas, melanomas and glioblastomas, inflammatory disorders such as asthma, septic shock, rheumatoid arthritis, psoriasis, inflammatory bowl syndrome and any other disease that responds to modulation of MAP kinase expression.
• Troglitazone, insulin, and PTP-1B modulators are non-limiting examples of pharmaceutical agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention for treating obesity and diabetes. The use of radiation treatments and chemotherapeutics such as Gemcytabine and cyclophosphamide are non-limiting examples of chemotherapeutic agents that can also be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention for oncology therapeutic applications. Those skilled in the art will recognize that other anti-cancer compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. siNA molecules) and are hence within the scope of the instant invention. Such compounds and therapies are well known in the art (see for exampleCancer: Principles and Practice of Oncology,Volumes 1 and 2, eds Devita, V. T., Hellman, S., and Rosenberg, S. A., J.B. Lippincott Company, Philadelphia, USA; incorporated herein by reference) and include, without limitations, folates, antifolates, pyrimidine analogs, fluoropyrimidines, purine analogs, adenosine analogs, topoisomerase I inhibitors, anthrapyrazoles, retinoids, antibiotics, anthacyclins, platinum analogs, alkylating agents, nitrosoureas, plant derived compounds such as vinca alkaloids, epipodophyllotoxins, tyrosine kinase inhibitors, taxols, radiation therapy, surgery, nutritional supplements, gene therapy, radiotherapy, for example 3D-CRT, immunotoxin therapy, for example ricin, and monoclonal antibodies. Specific examples of chemotherapeutic compounds that can be combined with or used in conjunction with the nucleic acid molecules of the invention include, but are not limited to, Paclitaxel; Docetaxel; Methotrexate; Doxorubin; Edatrexate; Vinorelbine; Tamoxifen; Leucovorin; 5-fluoro uridine (5-FU); Ionotecan; Cisplatin; Carboplatin; Amsacrine; Cytarabine; Bleomycin; Mitomycin C; Dactinomycin; Mithramycin; Hexamethylmelamine; Dacarbazine; L-asperginase; Nitrogen mustard; Melphalan, Chlorambucil; Busulfan; Ifosfamide; 4-hydroperoxycyclophosphamide, Thiotepa; Irinotecan (CAMPTOSAR®, CPT-11, Camptothecin-11, Campto) Tamoxifen, Herceptin; IMC C225; ABX-EGF: and combinations thereof are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA) of the instant invention. In addition, treatment of HCV infected subjects with siNA molecules of the invention targeting c-JUN or other MAP kinases involved in the maintenance or development of hepatocellular carcinoma can be combined with anti-viral compounds, such as siNA molecules targeting HCV RNA or other antiviral compounds known in the art (e.g., interferons, nucleoside analogs etc.). Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g., siNA molecules) are hence within the scope of the instant invention.
Example 12Diagnostic Uses
• The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates. siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one ca MAP nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).
• In a specific example, siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules (i.e., those that cleave only wild-type forms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules (i.e., those that cleave only mutant forms of target RNA) are used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
• All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
• 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.
• It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying siNA molecules with improved RNAi activity.
• The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.
• In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

• TABLE I
  MAP kinase Accession Numbers

  NM_002745	Homo sapiensmitogen-activated protein kinase 1 (MAPK1),transcript variant 1, mRNA.
  NM_138957	Homo sapiensmitogen-activated protein kinase 1 (MAPK1),transcript variant 2, mRNA.
  X60188	Human ERK1 mRNA for protein serine/threonine kinase (MAPK3).
  XM_055766	Homo sapiensmitogen-activated protein kinase 3 (MAPK3), mRNA
  NM_002747	Homo sapiensmitogen-activated protein kinase 4 (MAPK4), mRNA
  XM_165662	Homo sapiensMitogen-activated protein kinase 4 (Extracellular signal-regulated kinase 4) (ERK-4) (MAP kinase
  	isoform p63) (p63-MAPK) (LOC220131), mRNA
  NM_002748	Homo sapiensmitogen-activated protein kinase 6 (MAPK6), mRNA.
  XM_166057	Homo sapiensMitogen-activated protein kinase 6 (Extracellular signal-regulated kinase 3) (ERK-3) (MAP kinase
  	isoform p97) (p97-MAPK) (LOC220839), mRNA
  XM_035575	Homo sapiensmitogen-activated protein kinase 6 (MAPK6), mRNA
  NM_139033	Homo sapiensmitogen-activated protein kinase 7 (MAPK7),transcript variant 1, mRNA
  NM_139032	Homo sapiensmitogen-activated protein kinase 7 (MAPK7),transcript variant 2, mRNA
  NM_002749	Homo sapiensmitogen-activated protein kinase 7 (MAPK7),transcript variant 3, mRNA
  NM_139034	Homo sapiensmitogen-activated protein kinase 7 (MAPK7), transcript variant 4, mRNA
  NM_139049	Homo sapiensmitogen-activated protein kinase 8 (MAPK8),transcript variant 1, mRNA.
  NM_002750	Homo sapiensmitogen-activated protein kinase 8 (MAPK8),transcript variant 2, mRNA.
  NM_139046	Homo sapiensmitogen-activated protein kinase 8 (MAPK8),transcript variant 3, mRNA.
  NM_139047	Homo sapiensmitogen-activated protein kinase 8 (MAPK8), transcript variant 4, mRNA.
  NM_002752	Homo sapiensmitogen-activated protein kinase 9 (MAPK9),transcript variant 1, mRNA.
  NM_139068	Homo sapiensmitogen-activated protein kinase 9 (MAPK9),transcript variant 2, mRNA.
  NM_139069	Homo sapiensmitogen-activated protein kinase 9 (MAPK9),transcript variant 3, mRNA.
  NM_139070	Homo sapiensmitogen-activated protein kinase 9 (MAPK9), transcript variant 4, mRNA.
  NM_002753	Homo sapiensmitogen-activated protein kinase 10 (MAPK10),transcript variant 1, mRNA
  NM_138982	Homo sapiensmitogen-activated protein kinase 10 (MAPK10),transcript variant 2, mRNA
  NM_138980	Homo sapiensmitogen-activated protein kinase 10 (MAPK10),transcript variant 3, mRNA
  NM_138981	Homo sapiensmitogen-activated protein kinase 10 (MAPK10), transcript variant 4, mRNA
  NM_002751	Homo sapiensmitogen-activated protein kinase 11 (MAPK11),transcript variant 1, mRNA.
  NM_138993	Homo sapiensmitogen-activated protein kinase 11 (MAPK11),transcript variant 2, mRNA.
  NM_002969	Homo sapiensmitogen-activated protein kinase 12 (MAPK12), mRNA.
  NM_002754	Homo sapiensmitogen-activated protein kinase 13 (MAPK13), mRNA.
  NM_001315	Homo sapiensmitogen-activated protein kinase 14 (MAPK14),transcript variant 1, mRNA.
  NM_139012	Homo sapiensmitogen-activated protein kinase 14 (MAPK14),transcript variant 2, mRNA.
  NM_139013	Homo sapiensmitogen-activated protein kinase 14 (MAPK14),transcript variant 3, mRNA.
  NM_139014	Homo sapiensmitogen-activated protein kinase 14 (MAPK14), transcript variant 4, mRNA.
  NM_002755	Homo sapiensmitogen-activated protein kinase kinase 1 (MAP2K1), mRNA
  NM_030662	Homo sapiensmitogen-activated protein kinase kinase 2 (MAP2K2), mRNA
  NM_002756	Homo sapiensmitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant A, mRNA
  NM_145109	Homo sapiensmitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant B, mRNA
  NM_145110	Homo sapiensmitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant C, mRNA
  XM_008654	Homo sapiensmitogen-activated protein kinase kinase 4 (MAP2K4), mRNA
  NM_003010	Homo sapiensmitogen-activated protein kinase kinase 4 (MAP2K4), mRNA
  NM_145160	Homo sapiensmitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant A, mRNA
  NM_002757	Homo sapiensmitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant B, mRNA
  NM_145161	Homo sapiensmitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant C, mRNA
  NM_145162	Homo sapiensmitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant D, mRNA
  XM_113313	Homo sapiensmitogen-activated protein kinase kinase 6 (MAP2K6), mRNA
  NM_002758	Homo sapiensmitogen-activated protein kinase kinase 6 (MAP2K6), transcript variant 1, mRNA
  NM_031988	Homo sapiensmitogen-activated protein kinase kinase 6 (MAP2K6), transcript variant 2, mRNA
  NM_005043	Homo sapiensmitogen-activated protein kinase kinase 7 (MAP2K7), transcript variant A, mRNA
  NM_145185	Homo sapiensmitogen-activated protein kinase kinase 7 (MAP2K7), transcript variant B, mRNA
  NM_145329	Homo sapiensmitogen-activated protein kinase kinase 7 (MAP2K7), transcript variant C, mRNA
  AF042838	Homo sapiensmitogen-activated protein kinase kinase kinase 1 (MAP3K1), mRNA
  NM_006609	Homo sapiensmitogen-activated protein kinase kinase kinase 2 (MAP3K2), mRNA
  NM_002401	Homo sapiensmitogen-activated protein kinase kinase kinase 3 (MAP3K3), mRNA
  NM_005922	Homo sapiensmitogen-activated protein kinase kinase kinase 4 (MAP3K4), transcript variant 1, mRNA
  NM_006724	Homo sapiensmitogen-activated protein kinase kinase kinase 4 (MAP3K4), transcript variant 2, mRNA
  NM_005923	Homo sapiensmitogen-activated protein kinase kinase kinase 5 (MAP3K5), mRNA
  NM_004672	Homo sapiensmitogen-activated protein kinase kinase kinase 6 (MAP3K6), mRNA
  NM_003188	Homo sapiensmitogen-activated protein kinase kinase kinase 7 (MAP3K7), mRNA
  NM_005204	Homo sapiensmitogen-activated protein kinase kinase kinase 8 (MAP3K8), mRNA
  AF251442	Homo sapiensmitogen-activated protein kinase kinase kinase 9 (MAP3K9), mRNA
  NM_002446	Homo sapiensmitogen-activated protein kinase kinase kinase 10 (MAP3K10), mRNA
  NM_002419	Homo sapiensmitogen-activated protein kinase kinase kinase 11 (MAP3K11), mRNA
  NM_006301	Homo sapiensmitogen-activated protein kinase kinase kinase 12 (MAP3K12), mRNA
  NM_004721	Homo sapiensmitogen-activated protein kinase kinase kinase 13 (MAP3K13), mRNA
  NM_003954	Homo sapiensmitogen-activated protein kinase kinase kinase 14 (MAP3K14), mRNA
  NM_007181	Homo sapiensmitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), mRNA
  NM_004579	Homo sapiensmitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), mRNA
  NM_003618	Homo sapiensmitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3), mRNA
  NM_004834	Homo sapiensmitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), mRNA
  NM_006575	Homo sapiensmitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5), mRNA
  NM_003668	Homo sapiensmitogen-activated protein kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 1, mRNA
  NM_139078	Homo sapiensmitogen-activated protein kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 2, mRNA
  NM_004635	Homo sapiensmitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3), mRNA
  NM_004759	Homo sapiensmitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), transcript variant 1, mRNA
  NM_032960	Homo sapiensmitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), transcript variant 2, mRNA
  NM_005373	Homo sapiensmyeloproliferative leukemia virus oncogene (MPL), mRNA
  NM_016848	Homo sapiensneuronal Shc (SHC3), mRNA
  NM_002649	Homo sapiensphosphoinositide-3-kinase, catalytic, gamma polypeptide (PIK3CG), mRNA
  NM_021003	Homo sapiensprotein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform (PPM1A), mRNA
  NM_003942	Homo sapiensribosomal protein S6 kinase, 90 kD, polypeptide 4 (RPS6KA4), mRNA
  NM_004755	Homo sapiensribosomal protein S6 kinase, 90 kD, polypeptide 5 (RPS6KA5), mRNA
  NM_002228	Homo sapiensv-jun sarcoma virus 17 oncogene homolog (avian) (JUN), mRNA

• TABLE II
  MAP kinase siNA and Target Sequences

  		Seq			Seq			Seq
  Pos	Target Sequence	ID	UPos	Upper seq	ID	LPos	Lower seq	ID

  NM_002745 (MAPK1/ERK2)

  3	CCCUCCCUCCGCCCGCCCG	1	3	CCCUCCCUCCGCCCGCCCG	1	21	CGGGCGGGCGGAGGGAGGG	164
  21	GCCGGCCCGCCCGUCAGUC	2	21	GCCGGCCCGCCCGUCAGUC	2	39	GACUGACGGGCGGGCCGGC	165
  39	CUGGCAGGCAGGCAGGCAA	3	39	CUGGCAGGCAGGCAGGCAA	3	57	UUGCCUGCCUGCCUGCCAG	166
  57	AUCGGUCCGAGUGGCUGUC	4	57	AUCGGUCCGAGUGGCUGUC	4	75	GACAGCCACUCGGACCGAU	167
  75	CGGCUCUUCAGCUCUCCCG	5	75	CGGCUCUUCAGCUCUCCCG	5	93	CGGGAGAGCUGAAGAGCCG	168
  93	GCUCGGCGUCUUCCUUCCU	6	93	GCUCGGCGUCUUCCUUCCU	6	111	AGGAAGGAAGACGCCGAGC	169
  111	UCCUCCCGGUCAGCGUCGG	7	111	UCCUCCCGGUCAGCGUCGG	7	129	CCGACGCUGACCGGGAGGA	170
  129	GCGGCUGCACCGGCGGCGG	8	129	GCGGCUGCACCGGCGGCGG	8	147	CCGCCGCCGGUGCAGCCGC	171
  147	GCGCAGUCCCUGCGGGAGG	9	147	GCGCAGUCCCUGCGGGAGG	9	165	CCUCCCGCAGGGACUGCGC	172
  165	GGGCGACAAGAGCUGAGCG	10	165	GGGCGACAAGAGCUGAGCG	10	183	CGCUCAGCUCUUGUCGCCC	173
  183	GGCGGCCGCCGAGCGUCGA	11	183	GGCGGCCGCCGAGCGUCGA	11	201	UCGACGCUCGGCGGCCGCC	174
  201	AGCUCAGCGCGGCGGAGGC	12	201	AGCUCAGCGCGGCGGAGGC	12	219	GCCUCCGCCGCGCUGAGCU	175
  219	CGGCGGCGGCCCGGCAGCC	13	219	CGGCGGCGGCCCGGCAGCC	13	237	GGCUGCCGGGCCGCCGCCG	176
  237	CAACAUGGCGGCGGCGGCG	14	237	CAACAUGGCGGCGGCGGCG	14	255	CGCCGCCGCCGCCAUGUUG	177
  255	GGCGGCGGGCGCGGGCCCG	15	255	GGCGGCGGGCGCGGGCCCG	15	273	CGGGCCCGCGCCCGCCGCC	178
  273	GGAGAUGGUCCGCGGGCAG	16	273	GGAGAUGGUCCGCGGGCAG	16	291	CUGCCCGCGGACCAUCUCC	179
  291	GGUGUUCGACGUGGGGCCG	17	291	GGUGUUCGACGUGGGGCCG	17	309	CGGCCCCACGUCGAACACC	180
  309	GCGCUACACCAACCUCUCG	18	309	GCGCUACACCAACCUCUCG	18	327	CGAGAGGUUGGUGUAGCGC	181
  327	GUACAUCGGCGAGGGCGCC	19	327	GUACAUCGGCGAGGGCGCC	19	345	GGCGCCCUCGCCGAUGUAC	182
  345	CUACGGCAUGGUGUGCUCU	20	345	CUACGGCAUGGUGUGCUCU	20	363	AGAGCACACCAUGCCGUAG	183
  363	UGCUUAUGAUAAUGUCAAC	21	363	UGCUUAUGAUAAUGUCAAC	21	381	GUUGACAUUAUCAUAAGCA	184
  381	CAAAGUUCGAGUAGCUAUC	22	381	CAAAGUUCGAGUAGCUAUC	22	399	GAUAGCUACUCGAACUUUG	185
  399	CAAGAAAAUCAGCCCCUUU	23	399	CAAGAAAAUCAGCCCCUUU	23	417	AAAGGGGCUGAUUUUCUUG	186
  417	UGAGCACCAGACCUACUGC	24	417	UGAGCACCAGACCUACUGC	24	435	GCAGUAGGUCUGGUGCUCA	187
  435	CCAGAGAACCCUGAGGGAG	25	435	CCAGAGAACCCUGAGGGAG	25	453	CUCCCUCAGGGUUCUCUGG	188
  453	GAUAAAAAUCUUACUGCGC	26	453	GAUAAAAAUCUUACUGCGC	26	471	GCGCAGUAAGAUUUUUAUC	189
  471	CUUCAGACAUGAGAACAUC	27	471	CUUCAGACAUGAGAACAUC	27	489	GAUGUUCUCAUGUCUGAAG	190
  489	CAUUGGAAUCAAUGACAUU	28	489	CAUUGGAAUCAAUGACAUU	28	507	AAUGUCAUUGAUUCCAAUG	191
  507	UAUUCGAGCACCAACCAUC	29	507	UAUUCGAGCACCAACCAUC	29	525	GAUGGUUGGUGCUCGAAUA	192
  525	CGAGCAAAUGAAAGAUGUA	30	525	CGAGCAAAUGAAAGAUGUA	30	543	UACAUCUUUCAUUUGCUCG	193
  543	AUAUAUAGUACAGGACCUC	31	543	AUAUAUAGUACAGGACCUC	31	561	GAGGUCCUGUACUAUAUAU	194
  561	CAUGGAAACAGAUCUUUAC	32	561	CAUGGAAACAGAUCUUUAC	32	579	GUAAAGAUCUGUUUCCAUG	195
  579	CAAGCUCUUGAAGACACAA	33	579	CAAGCUCUUGAAGACACAA	33	597	UUGUGUCUUCAAGAGCUUG	196
  597	ACACCUCAGCAAUGACCAU	34	597	ACACCUCAGCAAUGACCAU	34	615	AUGGUCAUUGCUGAGGUGU	197
  615	UAUCUGCUAUUUUCUCUAC	35	615	UAUCUGCUAUUUUCUCUAC	35	633	GUAGAGAAAAUAGCAGAUA	198
  633	CCAGAUCCUCAGAGGGUUA	36	633	CCAGAUCCUCAGAGGGUUA	36	651	UAACCCUCUGAGGAUCUGG	199
  651	AAAAUAUAUCCAUUCAGCU	37	651	AAAAUAUAUCCAUUCAGCU	37	669	AGCUGAAUGGAUAUAUUUU	200
  669	UAACGUUCUGCACCGUGAC	38	669	UAACGUUCUGCACCGUGAC	38	687	GUCACGGUGCAGAACGUUA	201
  687	CCUCAAGCCUUCCAACCUG	39	687	CCUCAAGCCUUCCAACCUG	39	705	CAGGUUGGAAGGCUUGAGG	202
  705	GCUGCUCAACACCACCUGU	40	705	GCUGCUCAACACCACCUGU	40	723	ACAGGUGGUGUUGAGCAGC	203
  723	UGAUCUCAAGAUCUGUGAC	41	723	UGAUCUCAAGAUCUGUGAC	41	741	GUCACAGAUCUUGAGAUCA	204
  741	CUUUGGCCUGGCCCGUGUU	42	741	CUUUGGCCUGGCCCGUGUU	42	759	AACACGGGCCAGGCCAAAG	205
  759	UGCAGAUCCAGACCAUGAU	43	759	UGCAGAUCCAGACCAUGAU	43	777	AUCAUGGUCUGGAUCUGCA	206
  777	UCACACAGGGUUCCUGACA	44	777	UCACACAGGGUUCCUGACA	44	795	UGUCAGGAACCCUGUGUGA	207
  795	AGAAUAUGUGGCCACACGU	45	795	AGAAUAUGUGGCCACACGU	45	813	ACGUGUGGCCACAUAUUCU	208
  813	UUGGUACAGGGCUCCAGAA	46	813	UUGGUACAGGGCUCCAGAA	46	831	UUCUGGAGCCCUGUACCAA	209
  831	AAUUAUGUUGAAUUCCAAG	47	831	AAUUAUGUUGAAUUCCAAG	47	849	CUUGGAAUUCAACAUAAUU	210
  849	GGGCUACACCAAGUCCAUU	48	849	GGGCUACACCAAGUCCAUU	48	867	AAUGGACUUGGUGUAGCCC	211
  867	UGAUAUUUGGUCUGUAGGC	49	867	UGAUAUUUGGUCUGUAGGC	49	885	GCCUACAGACCAAAUAUCA	212
  885	CUGCAUUCUGGCAGAAAUG	50	885	CUGCAUUCUGGCAGAAAUG	50	903	CAUUUCUGCCAGAAUGCAG	213
  903	GCUUUCUAACAGGCCCAUC	51	903	GCUUUCUAACAGGCCCAUC	51	921	GAUGGGCCUGUUAGAAAGC	214
  921	CUUUCCAGGGAAGCAUUAU	52	921	CUUUCCAGGGAAGCAUUAU	52	939	AUAAUGCUUCCCUGGAAAG	215
  939	UCUUGACCAGCUGAAACAC	53	939	UCUUGACCAGCUGAAACAC	53	957	GUGUUUCAGCUGGUCAAGA	216
  957	CAUUUUGGGUAUUCUUGGA	54	957	CAUUUUGGGUAUUCUUGGA	54	975	UCCAAGAAUACCCAAAAUG	217
  975	AUCCCCAUCACAAGAAGAC	55	975	AUCCCCAUCACAAGAAGAC	55	993	GUCUUCUUGUGAUGGGGAU	218
  993	CCUGAAUUGUAUAAUAAAU	56	993	CCUGAAUUGUAUAAUAAAU	56	1011	AUUUAUUAUACAAUUCAGG	219
  1011	UUUAAAAGCUAGGAACUAU	57	1011	UUUAAAAGCUAGGAACUAU	57	1029	AUAGUUCCUAGCUUUUAAA	220
  1029	UUUGCUUUCUCUUCCACAC	58	1029	UUUGCUUUCUCUUCCACAC	58	1047	GUGUGGAAGAGAAAGCAAA	221
  1047	CAAAAAUAAGGUGCCAUGG	59	1047	CAAAAAUAAGGUGCCAUGG	59	1065	CCAUGGCACCUUAUUUUUG	222
  1065	GAACAGGCUGUUCCCAAAU	60	1065	GAACAGGCUGUUCCCAAAU	60	1083	AUUUGGGAACAGCCUGUUC	223
  1083	UGCUGACUCCAAAGCUCUG	61	1083	UGCUGACUCCAAAGCUCUG	61	1101	CAGAGCUUUGGAGUCAGCA	224
  1101	GGACUUAUUGGACAAAAUG	62	1101	GGACUUAUUGGACAAAAUG	62	1119	CAUUUUGUCCAAUAAGUCC	225
  1119	GUUGACAUUCAACCCACAC	63	1119	GUUGACAUUCAACCCACAC	63	1137	GUGUGGGUUGAAUGUCAAC	226
  1137	CAAGAGGAUUGAAGUAGAA	64	1137	CAAGAGGAUUGAAGUAGAA	64	1155	UUCUACUUCAAUCCUCUUG	227
  1155	ACAGGCUCUGGCCCACCCA	65	1155	ACAGGCUCUGGCCCACCCA	65	1173	UGGGUGGGCCAGAGCCUGU	228
  1173	AUAUCUGGAGCAGUAUUAC	66	1173	AUAUCUGGAGCAGUAUUAC	66	1191	GUAAUACUGCUCCAGAUAU	229
  1191	CGACCCGAGUGACGAGCCC	67	1191	CGACCCGAGUGACGAGCCC	67	1209	GGGCUCGUCACUCGGGUCG	230
  1209	CAUCGCCGAAGCACCAUUC	68	1209	CAUCGCCGAAGCACCAUUC	68	1227	GAAUGGUGCUUCGGCGAUG	231
  1227	CAAGUUCGACAUGGAAUUG	69	1227	CAAGUUCGACAUGGAAUUG	69	1245	CAAUUCCAUGUCGAACUUG	232
  1245	GGAUGACUUGCCUAAGGAA	70	1245	GGAUGACUUGCCUAAGGAA	70	1263	UUCCUUAGGCAAGUCAUCC	233
  1263	AAAGCUCAAAGAACUAAUU	71	1263	AAAGCUCAAAGAACUAAUU	71	1281	AAUUAGUUCUUUGAGCUUU	234
  1281	UUUUGAAGAGACUGCUAGA	72	1281	UUUUGAAGAGACUGCUAGA	72	1299	UCUAGCAGUCUCUUCAAAA	235
  1299	AUUCCAGCCAGGAUACAGA	73	1299	AUUCCAGCCAGGAUACAGA	73	1317	UCUGUAUCCUGGCUGGAAU	236
  1317	AUCUUAAAUUUGUCAGGAC	74	1317	AUCUUAAAUUUGUCAGGAC	74	1335	GUCCUGACAAAUUUAAGAU	237
  1335	CAAGGGCUCAGAGGACUGG	75	1335	CAAGGGCUCAGAGGACUGG	75	1353	CCAGUCCUCUGAGCCCUUG	238
  1353	GACGUGCUCAGACAUCGGU	76	1353	GACGUGCUCAGACAUCGGU	76	1371	ACCGAUGUCUGAGCACGUC	239
  1371	UGUUCUUCUUCCCAGUUCU	77	1371	UGUUCUUCUUCCCAGUUCU	77	1389	AGAACUGGGAAGAAGAACA	240
  1389	UUGACCCCUGGUCCUGUCU	78	1389	UUGACCCCUGGUCCUGUCU	78	1407	AGACAGGACCAGGGGUCAA	241
  1407	UCCAGCCCGUCUUGGCUUA	79	1407	UCCAGCCCGUCUUGGCUUA	79	1425	UAAGCCAAGACGGGCUGGA	242
  1425	AUCCACUUUGACUCCUUUG	80	1425	AUCCACUUUGACUCCUUUG	80	1443	CAAAGGAGUCAAAGUGGAU	243
  1443	GAGCCGUUUGGAGGGGCGG	81	1443	GAGCCGUUUGGAGGGGCGG	81	1461	CCGCCCCUCCAAACGGCUC	244
  1461	GUUUCUGGUAGUUGUGGCU	82	1461	GUUUCUGGUAGUUGUGGCU	82	1479	AGCCACAACUACCAGAAAC	245
  1479	UUUUAUGCUUUCAAAGAAU	83	1479	UUUUAUGCUUUCAAAGAAU	83	1497	AUUCUUUGAAAGCAUAAAA	246
  1497	UUUCUUCAGUCCAGAGAAU	84	1497	UUUCUUCAGUCCAGAGAAU	84	1515	AUUCUCUGGACUGAAGAAA	247
  1515	UUCCUCCUGGCAGCCCUGU	85	1515	UUCCUCCUGGCAGCCCUGU	85	1533	ACAGGGCUGCCAGGAGGAA	248
  1533	UGUGUGUCACCCAUUGGUG	86	1533	UGUGUGUCACCCAUUGGUG	86	1551	CACCAAUGGGUGACACACA	249
  1551	GACCUGCGGCAGUAUGUAC	87	1551	GACCUGCGGCAGUAUGUAC	87	1569	GUACAUACUGCCGCAGGUC	250
  1569	CUUCAGUGCACCUUACUGC	88	1569	CUUCAGUGCACCUUACUGC	88	1587	GCAGUAAGGUGCACUGAAG	251
  1587	CUUACUGUUGCUUUAGUCA	89	1587	CUUACUGUUGCUUUAGUCA	89	1605	UGACUAAAGCAACAGUAAG	252
  1605	ACUAAUUGCUUUCUGGUUU	90	1605	ACUAAUUGCUUUCUGGUUU	90	1623	AAACCAGAAAGCAAUUAGU	253
  1623	UGAAAGAUGCAGUGGUUCC	91	1623	UGAAAGAUGCAGUGGUUCC	91	1641	GGAACCACUGCAUCUUUCA	254
  1641	CUCCCUCUCCUGAAUCCUU	92	1641	CUCCCUCUCCUGAAUCCUU	92	1659	AAGGAUUCAGGAGAGGGAG	255
  1659	UUUCUACAUGAUGCCCUGC	93	1659	UUUCUACAUGAUGCCCUGC	93	1677	GCAGGGCAUCAUGUAGAAA	256
  1677	CUGACCAUGCAGCCGCACC	94	1677	CUGACCAUGCAGCCGCACC	94	1695	GGUGCGGCUGCAUGGUCAG	257
  1695	CAGAGAGAGAUUCUUCCCC	95	1695	CAGAGAGAGAUUCUUCCCC	95	1713	GGGGAAGAAUCUCUCUCUG	258
  1713	CAAUUGGCUCUAGUCACUG	96	1713	CAAUUGGCUCUAGUCACUG	96	1731	CAGUGACUAGAGCCAAUUG	259
  1731	GGCAUCUCACUUUAUGAUA	97	1731	GGCAUCUCACUUUAUGAUA	97	1749	UAUCAUAAAGUGAGAUGCC	260
  1749	AGGGAAGGCUACUACCUAG	98	1749	AGGGAAGGCUACUACCUAG	98	1767	CUAGGUAGUAGCCUUCCCU	261
  1767	GGGCACUUUAAGUCAGUGA	99	1767	GGGCACUUUAAGUCAGUGA	99	1785	UCACUGACUUAAAGUGCCC	262
  1785	ACAGCCCCUUAUUUGCACU	100	1785	ACAGCCCCUUAUUUGCACU	100	1803	AGUGCAAAUAAGGGGCUGU	263
  1803	UUCACCUUUUGACCAUAAC	101	1803	UUCACCUUUUGACCAUAAC	101	1821	GUUAUGGUCAAAAGGUGAA	264
  1821	CUGUUUCCCCAGAGCAGGA	102	1821	CUGUUUCCCCAGAGCAGGA	102	1839	UCCUGCUCUGGGGAAACAG	265
  1839	AGCUUGUGGAAAUACCUUG	103	1839	AGCUUGUGGAAAUACCUUG	103	1857	CAAGGUAUUUCCACAAGCU	266
  1857	GGCUGAUGUUGCAGCCUGC	104	1857	GGCUGAUGUUGCAGCCUGC	104	1875	GCAGGCUGCAACAUCAGCC	267
  1875	CAGCAAGUGCUUCCGUCUC	105	1875	CAGCAAGUGCUUCCGUCUC	105	1893	GAGACGGAAGCACUUGCUG	268
  1893	CCGGAAUCCUUGGGGAGCA	106	1893	CCGGAAUCCUUGGGGAGCA	106	1911	UGCUCCCCAAGGAUUCCGG	269
  1911	ACUUGUCCACGUCUUUUCU	107	1911	ACUUGUCCACGUCUUUUCU	107	1929	AGAAAAGACGUGGACAAGU	270
  1929	UCAUAUCAUGGUAGUCACU	108	1929	UCAUAUCAUGGUAGUCACU	108	1947	AGUGACUACCAUGAUAUGA	271
  1947	UAACAUAUAUAAGGUAUGU	109	1947	UAACAUAUAUAAGGUAUGU	109	1965	ACAUACCUUAUAUAUGUUA	272
  1965	UGCUAUUGGCCCAGCUUUU	110	1965	UGCUAUUGGCCCAGCUUUU	110	1983	AAAAGCUGGGCCAAUAGCA	273
  1983	UAGAAAAUGCAGUCAUUUU	111	1983	UAGAAAAUGCAGUCAUUUU	111	2001	AAAAUGACUGCAUUUUCUA	274
  2001	UUCUAAAUAAAAAGGAAGU	112	2001	UUCUAAAUAAAAAGGAAGU	112	2019	ACUUCCUUUUUAUUUAGAA	275
  2019	UACUGCACCCAGCAGUGUC	113	2019	UACUGCACCCAGCAGUGUC	113	2037	GACACUGCUGGGUGCAGUA	276
  2037	CACUCUGUAGUUACUGUGG	114	2037	CACUCUGUAGUUACUGUGG	114	2055	CCACAGUAACUACAGAGUG	277
  2055	GUCACUUGUACCAUAUAGA	115	2055	GUCACUUGUACCAUAUAGA	115	2073	UCUAUAUGGUACAAGUGAC	278
  2073	AGGUGUAACACUUGUCAAG	116	2073	AGGUGUAACACUUGUCAAG	116	2091	CUUGACAAGUGUUACACCU	279
  2091	GAAGCGUUAUGUGCAGUAC	117	2091	GAAGCGUUAUGUGCAGUAC	117	2109	GUACUGCACAUAACGCUUC	280
  2109	CUUAAUGUUUGUAAGACUU	118	2109	CUUAAUGUUUGUAAGACUU	118	2127	AAGUCUUACAAACAUUAAG	281
  2127	UACAAAAAAAGAUUUAAAG	119	2127	UACAAAAAAAGAUUUAAAG	119	2145	CUUUAAAUCUUUUUUUGUA	282
  2145	GUGGCAGCUUCACUCGACA	120	2145	GUGGCAGCUUCACUCGACA	120	2163	UGUCGAGUGAAGCUGCCAC	283
  2163	AUUUGGUGAGAGAAGUACA	121	2163	AUUUGGUGAGAGAAGUACA	121	2181	UGUACUUCUCUCACCAAAU	284
  2181	AAAGGUUGCAGUGCUGAGC	122	2181	AAAGGUUGCAGUGCUGAGC	122	2199	GCUCAGCACUGCAACCUUU	285
  2199	CUGUGGGCGGUUUCUGGGG	123	2199	CUGUGGGCGGUUUCUGGGG	123	2217	CCCCAGAAACCGCCCACAG	286
  2217	GAUGUCCCAGGGUGGAACU	124	2217	GAUGUCCCAGGGUGGAACU	124	2235	AGUUCCACCCUGGGACAUC	287
  2235	UCCACAUGCUGGUGCAUAU	125	2235	UCCACAUGCUGGUGCAUAU	125	2253	AUAUGCACCAGCAUGUGGA	288
  2253	UACGCCCUUGAGCUACUUC	126	2253	UACGCCCUUGAGCUACUUC	126	2271	GAAGUAGCUCAAGGGCGUA	289
  2271	CAAAUGUGGUUUAUACCUC	127	2271	CAAAUGUGGUUUAUACCUC	127	2289	GAGGUAUAAACCACAUUUG	290
  2289	CGCAGAUACAAGAAUCUUU	128	2289	CGCAGAUACAAGAAUCUUU	128	2307	AAAGAUUCUUGUAUCUGCG	291
  2307	UAUGAAUAUACAAUUCUUU	129	2307	UAUGAAUAUACAAUUCUUU	129	2325	AAAGAAUUGUAUAUUCAUA	292
  2325	UUUCCUUCUACAGCUUAGC	130	2325	UUUCCUUCUACAGCUUAGC	130	2343	GCUAAGCUGUAGAAGGAAA	293
  2343	CUCCGUCUUUUCAACCACG	131	2343	CUCCGUCUUUUCAACCACG	131	2361	CGUGGUUGAAAAGACGGAG	294
  2361	GAACAUUUAAAACCCGACC	132	2361	GAACAUUUAAAACCCGACC	132	2379	GGUCGGGUUUUAAAUGUUC	295
  2379	CUACUAGCACUGUUCUGUC	133	2379	CUACUAGCACUGUUCUGUC	133	2397	GACAGAACAGUGCUAGUAG	296
  2397	CCUCAAGUACUCAAAUAUU	134	2397	CCUCAAGUACUCAAAUAUU	134	2415	AAUAUUUGAGUACUUGAGG	297
  2415	UUCUGAUACUGCUGAGUCA	135	2415	UUCUGAUACUGCUGAGUCA	135	2433	UGACUCAGCAGUAUCAGAA	298
  2433	AGACUGUCAGAAAAAGCUA	136	2433	AGACUGUCAGAAAAAGCUA	136	2451	UAGCUUUUUCUGACAGUCU	299
  2451	AGCACUAACUCGUGUUUGG	137	2451	AGCACUAACUCGUGUUUGG	137	2469	CCAAACACGAGUUAGUGCU	300
  2469	GAGCUCUAUCCAUAUUUUA	138	2469	GAGCUCUAUCCAUAUUUUA	138	2487	UAAAAUAUGGAUAGAGCUC	301
  2487	ACUGAUCUCUUUAAGUAUU	139	2487	ACUGAUCUCUUUAAGUAUU	139	2505	AAUACUUAAAGAGAUCAGU	302
  2505	UUGUUCCUGCCACUGUGUA	140	2505	UUGUUCCUGCCACUGUGUA	140	2523	UACACAGUGGCAGGAACAA	303
  2523	ACUGUGGAGUUGACUCGGU	141	2523	ACUGUGGAGUUGACUCGGU	141	2541	ACCGAGUCAACUCCACAGU	304
  2541	UGUUCUGUCCCAGUGCGGU	142	2541	UGUUCUGUCCCAGUGCGGU	142	2559	ACCGCACUGGGACAGAACA	305
  2559	UGCCUCCUCUUGACUUCCC	143	2559	UGCCUCCUCUUGACUUCCC	143	2577	GGGAAGUCAAGAGGAGGCA	306
  2577	CCACUGCUCUCUGUGGUGA	144	2577	CCACUGCUCUCUGUGGUGA	144	2595	UCACCACAGAGAGCAGUGG	307
  2595	AGAAAUUUGCCUUGUUCAA	145	2595	AGAAAUUUGCCUUGUUCAA	145	2613	UUGAACAAGGCAAAUUUCU	308
  2613	AUAAUUACUGUACCCUCGC	146	2613	AUAAUUACUGUACCCUCGC	146	2631	GCGAGGGUACAGUAAUUAU	309
  2631	CAUGACUGUUACAGCUUUC	147	2631	CAUGACUGUUACAGCUUUC	147	2649	GAAAGCUGUAACAGUCAUG	310
  2649	CUGUGCAGAGAUGACUGUC	148	2649	CUGUGCAGAGAUGACUGUC	148	2667	GACAGUCAUCUCUGCACAG	311
  2667	CCAAGUGCCACAUGCCUAC	149	2667	CCAAGUGCCACAUGCCUAC	149	2685	GUAGGCAUGUGGCACUUGG	312
  2685	CGAUUGAAAUGAAAACUCU	150	2685	CGAUUGAAAUGAAAACUCU	150	2703	AGAGUUUUCAUUUCAAUCG	313
  2703	UAUUGUUACCUCUGAGUUG	151	2703	UAUUGUUACCUCUGAGUUG	151	2721	CAACUCAGAGGUAACAAUA	314
  2721	GUGUUCCACGGAAAAUGCU	152	2721	GUGUUCCACGGAAAAUGCU	152	2739	AGCAUUUUCCGUGGAACAC	315
  2739	UAUCCAGCAGAUCAUUUAG	153	2739	UAUCCAGCAGAUCAUUUAG	153	2757	CUAAAUGAUCUGCUGGAUA	316
  2757	GGAAAAAUAAUUCUAUUUU	154	2757	GGAAAAAUAAUUCUAUUUU	154	2775	AAAAUAGAAUUAUUUUUCC	317
  2775	UUAGCUUUUCAUUUCUCAG	155	2775	UUAGCUUUUCAUUUCUCAG	155	2793	CUGAGAAAUGAAAAGCUAA	318
  2793	GCUGUCCUUUUUUCUUGUU	156	2793	GCUGUCCUUUUUUCUUGUU	156	2811	AACAAGAAAAAAGGACAGC	319
  2811	UUGAUUUUUGACAGCAAUG	157	2811	UUGAUUUUUGACAGCAAUG	157	2829	CAUUGCUGUCAAAAAUCAA	320
  2829	GGAGAAUGGGUUAUAUAAA	158	2829	GGAGAAUGGGUUAUAUAAA	158	2847	UUUAUAUAACCCAUUCUCC	321
  2847	AGACUGCCUGCUAAUAUGA	159	2847	AGACUGCCUGCUAAUAUGA	159	2865	UCAUAUUAGCAGGCAGUCU	322
  2865	AACAGAAAUGCAUUUGUAA	160	2865	AACAGAAAUGCAUUUGUAA	160	2883	UUACAAAUGCAUUUCUGUU	323
  2883	AUUCAUGAAAAUAAAUGUA	161	2883	AUUCAUGAAAAUAAAUGUA	161	2901	UACAUUUAUUUUCAUGAAU	324
  2901	ACAUCUUCUAUCUUCAAAA	162	2901	ACAUCUUCUAUCUUCAAAA	162	2919	UUUUGAAGAUAGAAGAUGU	325
  2913	UUCAAAAAAAAAAAAAAAA	163	2913	UUCAAAAAAAAAAAAAAAA	163	2931	UUUUUUUUUUUUUUUUGAA	326

  		Seq			Seq			Seq
  Pos	Target Sequence	ID	UPos	Upper seq	ID	LPos	Lower seq	ID

  XM_055766.6 (MAPK3/ERK1)

  3	CGGGGCCUCGGGCGGGGCC	327	3	CGGGGCCUCGGGCGGGGCC	327	21	GGCCCCGCCCGAGGCCCCG	432
  21	CGCCGUGGGGAGGAGGGCG	328	21	CGCCGUGGGGAGGAGGGCG	328	39	CGCCCUCCUCCCCACGGCG	433
  39	GGUGGGAGGGGAGGAGUGG	329	39	GGUGGGAGGGGAGGAGUGG	329	57	CCACUCCUCCCCUCCCACC	434
  57	GAGAUGGCGGCGGCGGCGG	330	57	GAGAUGGCGGCGGCGGCGG	330	75	CCGCCGCCGCCGCCAUCUC	435
  75	GCUCAGGGGGGCGGGGGCG	331	75	GCUCAGGGGGGCGGGGGCG	331	93	CGCCCCCGCCCCCCUGAGC	436
  93	GGGGAGCCCCGUAGAACCG	332	93	GGGGAGCCCCGUAGAACCG	332	111	CGGUUCUACGGGGCUCCCC	437
  111	GAGGGGGUCGGCCCGGGGG	333	111	GAGGGGGUCGGCCCGGGGG	333	129	CCCCCGGGCCGACCCCCUC	438
  129	GUCCCGGGGGAGGUGGAGA	334	129	GUCCCGGGGGAGGUGGAGA	334	147	UCUCCACCUCCCCCGGGAC	439
  147	AUGGUGAAGGGGCAGCCGU	335	147	AUGGUGAAGGGGCAGCCGU	335	165	ACGGCUGCCCCUUCACCAU	440
  165	UUCGACGUGGGCCCGCGCU	336	165	UUCGACGUGGGCCCGCGCU	336	183	AGCGCGGGCCCACGUCGAA	441
  183	UACACGCAGUUGCAGUACA	337	183	UACACGCAGUUGCAGUACA	337	201	UGUACUGCAACUGCGUGUA	442
  201	AUCGGCGAGGGCGCGUACG	338	201	AUCGGCGAGGGCGCGUACG	338	219	CGUACGCGCCCUCGCCGAU	443
  219	GGCAUGGUCAGCUCGGCCU	339	219	GGCAUGGUCAGCUCGGCCU	339	237	AGGCCGAGCUGACCAUGCC	444
  237	UAUGACCACGUGCGCAAGA	340	237	UAUGACCACGUGCGCAAGA	340	255	UCUUGCGCACGUGGUCAUA	445
  255	ACUCGCGUGGCCAUCAAGA	341	255	ACUCGCGUGGCCAUCAAGA	341	273	UCUUGAUGGCCACGCGAGU	446
  273	AAGAUCAGCCCCUUCGAAC	342	273	AAGAUCAGCCCCUUCGAAC	342	291	GUUCGAAGGGGCUGAUCUU	447
  291	CAUCAGACCUACUGCCAGC	343	291	CAUCAGACCUACUGCCAGC	343	309	GCUGGCAGUAGGUCUGAUG	448
  309	CGCACGCUCCGGGAGAUCC	344	309	CGCACGCUCCGGGAGAUCC	344	327	GGAUCUCCCGGAGCGUGCG	449
  327	CAGAUCCUGCUGCGCUUCC	345	327	CAGAUCCUGCUGCGCUUCC	345	345	GGAAGCGCAGCAGGAUCUG	450
  345	CGCCAUGAGAAUGUCAUCG	346	345	CGCCAUGAGAAUGUCAUCG	346	363	CGAUGACAUUCUCAUGGCG	451
  363	GGCAUCCGAGACAUUCUGC	347	363	GGCAUCCGAGACAUUCUGC	347	381	GCAGAAUGUCUCGGAUGCC	452
  381	CGGGCGUCCACCCUGGAAG	348	381	CGGGCGUCCACCCUGGAAG	348	399	CUUCCAGGGUGGACGCCCG	453
  399	GCCAUGAGAGAUGUCUACA	349	399	GCCAUGAGAGAUGUCUACA	349	417	UGUAGACAUCUCUCAUGGC	454
  417	AUUGUGCAGGACCUGAUGG	350	417	AUUGUGCAGGACCUGAUGG	350	435	CCAUCAGGUCCUGCACAAU	455
  435	GAGACUGACCUGUACAAGU	351	435	GAGACUGACCUGUACAAGU	351	453	ACUUGUACAGGUCAGUCUC	456
  453	UUGCUGAAAAGCCAGCAGC	352	453	UUGCUGAAAAGCCAGCAGC	352	471	GCUGCUGGCUUUUCAGCAA	457
  471	CUGAGCAAUGACCAUAUCU	353	471	CUGAGCAAUGACCAUAUCU	353	489	AGAUAUGGUCAUUGCUCAG	458
  489	UGCUACUUCCUCUACCAGA	354	489	UGCUACUUCCUCUACCAGA	354	507	UCUGGUAGAGGAAGUAGCA	459
  507	AUCCUGCGGGGCCUCAAGU	355	507	AUCCUGCGGGGCCUCAAGU	355	525	ACUUGAGGCCCCGCAGGAU	460
  525	UACAUCCACUCCGCCAACG	356	525	UACAUCCACUCCGCCAACG	356	543	CGUUGGCGGAGUGGAUGUA	461
  543	GUGCUCCACCGAGAUCUAA	357	543	GUGCUCCACCGAGAUCUAA	357	561	UUAGAUCUCGGUGGAGCAC	462
  561	AAGCCCUCCAACCUGCUCA	358	561	AAGCCCUCCAACCUGCUCA	358	579	UGAGCAGGUUGGAGGGCUU	463
  579	AUCAACACCACCUGCGACC	359	579	AUCAACACCACCUGCGACC	359	597	GGUCGCAGGUGGUGUUGAU	464
  597	CUUAAGAUUUGUGAUUUCG	360	597	CUUAAGAUUUGUGAUUUCG	360	615	CGAAAUCACAAAUCUUAAG	465
  615	GGCCUGGCCCGGAUUGCCG	361	615	GGCCUGGCCCGGAUUGCCG	361	633	CGGCAAUCCGGGCCAGGCC	466
  633	GAUCCUGAGCAUGACCACA	362	633	GAUCCUGAGCAUGACCACA	362	651	UGUGGUCAUGCUCAGGAUC	467
  651	ACCGGCUUCCUGACGGAGU	363	651	ACCGGCUUCCUGACGGAGU	363	669	ACUCCGUCAGGAAGCCGGU	468
  669	UAUGUGGCUACGCGCUGGU	364	669	UAUGUGGCUACGCGCUGGU	364	687	ACCAGCGCGUAGCCACAUA	469
  687	UACCGGGCCCCAGAGAUCA	365	687	UACCGGGCCCCAGAGAUCA	365	705	UGAUCUCUGGGGCCCGGUA	470
  705	AUGCUGAACUCCAAGGGCU	366	705	AUGCUGAACUCCAAGGGCU	366	723	AGCCCUUGGAGUUCAGCAU	471
  723	UAUACCAAGUCCAUCGACA	367	723	UAUACCAAGUCCAUCGACA	367	741	UGUCGAUGGACUUGGUAUA	472
  741	AUCUGGUCUGUGGGCUGCA	368	741	AUCUGGUCUGUGGGCUGCA	368	759	UGCAGCCCACAGACCAGAU	473
  759	AUUCUGGCUGAGAUGCUCU	369	759	AUUCUGGCUGAGAUGCUCU	369	777	AGAGCAUCUCAGCCAGAAU	474
  777	UCUAACCGGCCCAUCUUCC	370	777	UCUAACCGGCCCAUCUUCC	370	795	GGAAGAUGGGCCGGUUAGA	475
  795	CCUGGCAAGCACUACCUGG	371	795	CCUGGCAAGCACUACCUGG	371	813	CCAGGUAGUGCUUGCCAGG	476
  813	GAUCAGCUCAACCACAUUC	372	813	GAUCAGCUCAACCACAUUC	372	831	GAAUGUGGUUGAGCUGAUC	477
  831	CUGGGCAUCCUGGGCUCCC	373	831	CUGGGCAUCCUGGGCUCCC	373	849	GGGAGCCCAGGAUGCCCAG	478
  849	CCAUCCCAGGAGGACCUGA	374	849	CCAUCCCAGGAGGACCUGA	374	867	UCAGGUCCUCCUGGGAUGG	479
  867	AAUUGUAUCAUCAACAUGA	375	867	AAUUGUAUCAUCAACAUGA	375	885	UCAUGUUGAUGAUACAAUU	480
  885	AAGGCCCGAAACUACCUAC	376	885	AAGGCCCGAAACUACCUAC	376	903	GUAGGUAGUUUCGGGCCUU	481
  903	CAGUCUCUGCCCUCCAAGA	377	903	CAGUCUCUGCCCUCCAAGA	377	921	UCUUGGAGGGCAGAGACUG	482
  921	ACCAAGGUGGCUUGGGCCA	378	921	ACCAAGGUGGCUUGGGCCA	378	939	UGGCCCAAGCCACCUUGGU	483
  939	AAGCUUUUCCCCAAGUCAG	379	939	AAGCUUUUCCCCAAGUCAG	379	957	CUGACUUGGGGAAAAGCUU	484
  957	GACUCCAAAGCCCUUGACC	380	957	GACUCCAAAGCCCUUGACC	380	975	GGUCAAGGGCUUUGGAGUC	485
  975	CUGCUGGACCGGAUGUUAA	381	975	CUGCUGGACCGGAUGUUAA	381	993	UUAACAUCCGGUCCAGCAG	486
  993	ACCUUUAACCCCAAUAAAC	382	993	ACCUUUAACCCCAAUAAAC	382	1011	GUUUAUUGGGGUUAAAGGU	487
  1011	CGGAUCACAGUGGAGGAAG	383	1011	CGGAUCACAGUGGAGGAAG	383	1029	CUUCCUCCACUGUGAUCCG	488
  1029	GCGCUGGCUCACCCCUACC	384	1029	GCGCUGGCUCACCCCUACC	384	1047	GGUAGGGGUGAGCCAGCGC	489
  1047	CUGGAGCAGUACUAUGACC	385	1047	CUGGAGCAGUACUAUGACC	385	1065	GGUCAUAGUACUGCUCCAG	490
  1065	CCGACGGAUGAGCCAGUGG	386	1065	CCGACGGAUGAGCCAGUGG	386	1083	CCACUGGCUCAUCCGUCGG	491
  1083	GCCGAGGAGCCCUUCACCU	387	1083	GCCGAGGAGCCCUUCACCU	387	1101	AGGUGAAGGGCUCCUCGGC	492
  1101	UUCGCCAUGGAGCUGGAUG	388	1101	UUCGCCAUGGAGCUGGAUG	388	1119	CAUCCAGCUCCAUGGCGAA	493
  1119	GACCUACCUAAGGAGCGGC	389	1119	GACCUACCUAAGGAGCGGC	389	1137	GCCGCUCCUUAGGUAGGUC	494
  1137	CUGAAGGAGCUCAUCUUCC	390	1137	CUGAAGGAGCUCAUCUUCC	390	1155	GGAAGAUGAGCUCCUUCAG	495
  1155	CAGGAGACAGCACGCUUCC	391	1155	CAGGAGACAGCACGCUUCC	391	1173	GGAAGCGUGCUGUCUCCUG	496
  1173	CAGCCCGGAGUGCUGGAGG	392	1173	CAGCCCGGAGUGCUGGAGG	392	1191	CCUCCAGCACUCCGGGCUG	497
  1191	GCCCCCUAGCCCAGACAGA	393	1191	GCCCCCUAGCCCAGACAGA	393	1209	UCUGUCUGGGCUAGGGGGC	498
  1209	ACAUCUCUGCACCCUGGGG	394	1209	ACAUCUCUGCACCCUGGGG	394	1227	CCCCAGGGUGCAGAGAUGU	499
  1227	GCCUGGAACAGAACUGGCA	395	1227	GCCUGGAACAGAACUGGCA	395	1245	UGCCAGUUCUGUUCCAGGC	500
  1245	AAAGAGGCAAGAGGUCACU	396	1245	AAAGAGGCAAGAGGUCACU	396	1263	AGUGACCUCUUGCCUCUUU	501
  1263	UGAGGGCCUCUGUCACCCA	397	1263	UGAGGGCCUCUGUCACCCA	397	1281	UGGGUGACAGAGGCCCUCA	502
  1281	AGGACCUGCCUCCUGCCUG	398	1281	AGGACCUGCCUCCUGCCUG	398	1299	CAGGCAGGAGGCAGGUCCU	503
  1299	GCCCCUCUCCCGCCAGACU	399	1299	GCCCCUCUCCCGCCAGACU	399	1317	AGUCUGGCGGGAGAGGGGC	504
  1317	UGUUAGAAAAUGGACACUG	400	1317	UGUUAGAAAAUGGACACUG	400	1335	CAGUGUCCAUUUUCUAACA	505
  1335	GUGCCCAGCCCGGACCUUG	401	1335	GUGCCCAGCCCGGACCUUG	401	1353	CAAGGUCCGGGCUGGGCAC	506
  1353	GGCAGCCCAGGCCGGGGUG	402	1353	GGCAGCCCAGGCCGGGGUG	402	1371	CACCCCGGCCUGGGCUGCC	507
  1371	GGAGCAUGGGCCUGGCCAC	403	1371	GGAGCAUGGGCCUGGCCAC	403	1389	GUGGCCAGGCCCAUGCUCC	508
  1389	CCUCUCUCCUUUGCUGAGG	404	1389	CCUCUCUCCUUUGCUGAGG	404	1407	CCUCAGCAAAGGAGAGAGG	509
  1407	GCCUCCAGCUUCAGGCAGG	405	1407	GCCUCCAGCUUCAGGCAGG	405	1425	CCUGCCUGAAGCUGGAGGC	510
  1425	GCCAAGGCCUUCUCCUCCC	406	1425	GCCAAGGCCUUCUCCUCCC	406	1443	GGGAGGAGAAGGCCUUGGC	511
  1443	CCACCCGCCCUCCCCACGG	407	1443	CCACCCGCCCUCCCCACGG	407	1461	CCGUGGGGAGGGCGGGUGG	512
  1461	GGGCCUCGGGACCUCAGGU	408	1461	GGGCCUCGGGACCUCAGGU	408	1479	ACCUGAGGUCCCGAGGCCC	513
  1479	UGGCCCCAGUUCAAUCUCC	409	1479	UGGCCCCAGUUCAAUCUCC	409	1497	GGAGAUUGAACUGGGGCCA	514
  1497	CCGCUGCUGCUGCUGCGCC	410	1497	CCGCUGCUGCUGCUGCGCC	410	1515	GGCGCAGCAGCAGCAGCGG	515
  1515	CCUUACCUUCCCCAGCGUC	411	1515	CCUUACCUUCCCCAGCGUC	411	1533	GACGCUGGGGAAGGUAAGG	516
  1533	CCCAGUCUCUGGCAGUUCU	412	1533	CCCAGUCUCUGGCAGUUCU	412	1551	AGAACUGCCAGAGACUGGG	517
  1551	UGGAAUGGAAGGGUUCUGG	413	1551	UGGAAUGGAAGGGUUCUGG	413	1569	CCAGAACCCUUCCAUUCCA	518
  1569	GCUGCCCCAACCUGCUGAA	414	1569	GCUGCCCCAACCUGCUGAA	414	1587	UUCAGCAGGUUGGGGCAGC	519
  1587	AGGGCAGAGGUGGAGGGUG	415	1587	AGGGCAGAGGUGGAGGGUG	415	1605	CACCCUCCACCUCUGCCCU	520
  1605	GGGGGGCGCUGAGUAGGGA	416	1605	GGGGGGCGCUGAGUAGGGA	416	1623	UCCCUACUCAGCGCCCCCC	521
  1623	ACUCAGGGCCAUGCCUGCC	417	1623	ACUCAGGGCCAUGCCUGCC	417	1641	GGCAGGCAUGGCCCUGAGU	522
  1641	CCCCCUCAUCUCAUUCAAA	418	1641	CCCCCUCAUCUCAUUCAAA	418	1659	UUUGAAUGAGAUGAGGGGG	523
  1659	ACCCCACCCUAGUUUCCCU	419	1659	ACCCCACCCUAGUUUCCCU	419	1677	AGGGAAACUAGGGUGGGGU	524
  1677	UGAAGGAACAUUCCUUAGU	420	1677	UGAAGGAACAUUCCUUAGU	420	1695	ACUAAGGAAUGUUCCUUCA	525
  1695	UCUCAAGGGCUAGCAUCCC	421	1695	UCUCAAGGGCUAGCAUCCC	421	1713	GGGAUGCUAGCCCUUGAGA	526
  1713	CUGAGGAGCCAGGCCGGGC	422	1713	CUGAGGAGCCAGGCCGGGC	422	1731	GCCCGGCCUGGCUCCUCAG	527
  1731	CCGAAUCCCCUCCCUGUCA	423	1731	CCGAAUCCCCUCCCUGUCA	423	1749	UGACAGGGAGGGGAUUCGG	528
  1749	AAAGCUGUCACUUCGCGUG	424	1749	AAAGCUGUCACUUCGCGUG	424	1767	CACGCGAAGUGACAGCUUU	529
  1767	GCCCUCGCUGCUUCUGUGU	425	1767	GCCCUCGCUGCUUCUGUGU	425	1785	ACACAGAAGCAGCGAGGGC	530
  1785	UGUGGUGAGCAGAAGUGGA	426	1785	UGUGGUGAGCAGAAGUGGA	426	1803	UCCACUUCUGCUCACCACA	531
  1803	AGCUGGGGGGCGUGGAGAG	427	1803	AGCUGGGGGGCGUGGAGAG	427	1821	CUCUCCACGCCCCCCAGCU	532
  1821	GCCCGGCGCCCCUGCCACC	428	1821	GCCCGGCGCCCCUGCCACC	428	1839	GGUGGCAGGGGCGCCGGGC	533
  1839	CUCCCUGACCCGUCUAAUA	429	1839	CUCCCUGACCCGUCUAAUA	429	1857	UAUUAGACGGGUCAGGGAG	534
  1857	AUAUAAAUAUAGAGAUGUG	430	1857	AUAUAAAUAUAGAGAUGUG	430	1875	CACAUCUCUAUAUUUAUAU	535
  1865	AUAGAGAUGUGUCUAUGGC	431	1865	AUAGAGAUGUGUCUAUGGC	431	1883	GCCAUAGACACAUCUCUAU	536

  		ID			Seq			Seq
  Pos	Target Sequence	Seq	UPos	Upper seq	ID	LPos	Lower seq	ID

  NM_002750 (MAPK8/JNK1)

  3	UAAUUGCUUGCCAUCAUGA	537	3	UAAUUGCUUGCCAUCAUGA	537	21	UCAUGAUGGCAAGCAAUUA	616
  21	AGCAGAAGCAAGCGUGACA	538	21	AGCAGAAGCAAGCGUGACA	538	39	UGUCACGCUUGCUUCUGCU	617
  39	AACAAUUUUUAUAGUGUAG	539	39	AACAAUUUUUAUAGUGUAG	539	57	CUACACUAUAAAAAUUGUU	618
  57	GAGAUUGGAGAUUCUACAU	540	57	GAGAUUGGAGAUUCUACAU	540	75	AUGUAGAAUCUCCAAUCUC	619
  75	UUCACAGUCCUGAAACGAU	541	75	UUCACAGUCCUGAAACGAU	541	93	AUCGUUUCAGGACUGUGAA	620
  93	UAUCAGAAUUUAAAACCUA	542	93	UAUCAGAAUUUAAAACCUA	542	111	UAGGUUUUAAAUUCUGAUA	621
  111	AUAGGCUCAGGAGCUCAAG	543	111	AUAGGCUCAGGAGCUCAAG	543	129	CUUGAGCUCCUGAGCCUAU	622
  129	GGAAUAGUAUGCGCAGCUU	544	129	GGAAUAGUAUGCGCAGCUU	544	147	AAGCUGCGCAUACUAUUCC	623
  147	UAUGAUGCCAUUCUUGAAA	545	147	UAUGAUGCCAUUCUUGAAA	545	165	UUUCAAGAAUGGCAUCAUA	624
  165	AGAAAUGUUGCAAUCAAGA	546	165	AGAAAUGUUGCAAUCAAGA	546	183	UCUUGAUUGCAACAUUUCU	625
  183	AAGCUAAGCCGACCAUUUC	547	183	AAGCUAAGCCGACCAUUUC	547	201	GAAAUGGUCGGCUUAGCUU	626
  201	CAGAAUCAGACUCAUGCCA	548	201	CAGAAUCAGACUCAUGCCA	548	219	UGGCAUGAGUCUGAUUCUG	627
  219	AAGCGGGCCUACAGAGAGC	549	219	AAGCGGGCCUACAGAGAGC	549	237	GCUCUCUGUAGGCCCGCUU	628
  237	CUAGUUCUUAUGAAAUGUG	550	237	CUAGUUCUUAUGAAAUGUG	550	255	CACAUUUCAUAAGAACUAG	629
  255	GUUAAUCACAAAAAUAUAA	551	255	GUUAAUCACAAAAAUAUAA	551	273	UUAUAUUUUUGUGAUUAAC	630
  273	AUUGGCCUUUUGAAUGUUU	552	273	AUUGGCCUUUUGAAUGUUU	552	291	AAACAUUCAAAAGGCCAAU	631
  291	UUCACACCACAGAAAUCCC	553	291	UUCACACCACAGAAAUCCC	553	309	GGGAUUUCUGUGGUGUGAA	632
  309	CUAGAAGAAUUUCAAGAUG	554	309	CUAGAAGAAUUUCAAGAUG	554	327	CAUCUUGAAAUUCUUCUAG	633
  327	GUUUACAUAGUCAUGGAGC	555	327	GUUUACAUAGUCAUGGAGC	555	345	GCUCCAUGACUAUGUAAAC	634
  345	CUCAUGGAUGCAAAUCUUU	556	345	CUCAUGGAUGCAAAUCUUU	556	363	AAAGAUUUGCAUCCAUGAG	635
  363	UGCCAAGUGAUUCAGAUGG	557	363	UGCCAAGUGAUUCAGAUGG	557	381	CCAUCUGAAUCACUUGGCA	636
  381	GAGCUAGAUCAUGAAAGAA	558	381	GAGCUAGAUCAUGAAAGAA	558	399	UUCUUUCAUGAUCUAGCUC	637
  399	AUGUCCUACCUUCUCUAUC	559	399	AUGUCCUACCUUCUCUAUC	559	417	GAUAGAGAAGGUAGGACAU	638
  417	CAGAUGCUGUGUGGAAUCA	560	417	CAGAUGCUGUGUGGAAUCA	560	435	UGAUUCCACACAGCAUCUG	639
  435	AAGCACCUUCAUUCUGCUG	561	435	AAGCACCUUCAUUCUGCUG	561	453	CAGCAGAAUGAAGGUGCUU	640
  453	GGAAUUAUUCAUCGGGACU	562	453	GGAAUUAUUCAUCGGGACU	562	471	AGUCCCGAUGAAUAAUUCC	641
  471	UUAAAGCCCAGUAAUAUAG	563	471	UUAAAGCCCAGUAAUAUAG	563	489	CUAUAUUACUGGGCUUUAA	642
  489	GUAGUAAAAUCUGAUUGCA	564	489	GUAGUAAAAUCUGAUUGCA	564	507	UGCAAUCAGAUUUUACUAC	643
  507	ACUUUGAAGAUUCUUGACU	565	507	ACUUUGAAGAUUCUUGACU	565	525	AGUCAAGAAUCUUCAAAGU	644
  525	UUCGGUCUGGCCAGGACUG	566	525	UUCGGUCUGGCCAGGACUG	566	543	CAGUCCUGGCCAGACCGAA	645
  543	GCAGGAACGAGUUUUAUGA	567	543	GCAGGAACGAGUUUUAUGA	567	561	UCAUAAAACUCGUUCCUGC	646
  561	AUGACGCCUUAUGUAGUGA	568	561	AUGACGCCUUAUGUAGUGA	568	579	UCACUACAUAAGGCGUCAU	647
  579	ACUCGCUACUACAGAGCAC	569	579	ACUCGCUACUACAGAGCAC	569	597	GUGCUCUGUAGUAGCGAGU	648
  597	CCCGAGGUCAUCCUUGGCA	570	597	CCCGAGGUCAUCCUUGGCA	570	615	UGCCAAGGAUGACCUCGGG	649
  615	AUGGGCUACAAGGAAAACG	571	615	AUGGGCUACAAGGAAAACG	571	633	CGUUUUCCUUGUAGCCCAU	650
  633	GUGGAUUUAUGGUCUGUGG	572	633	GUGGAUUUAUGGUCUGUGG	572	651	CCACAGACCAUAAAUCCAC	651
  651	GGGUGCAUUAUGGGAGAAA	573	651	GGGUGCAUUAUGGGAGAAA	573	669	UUUCUCCCAUAAUGCACCC	652
  669	AUGGUUUGCCACAAAAUCC	574	669	AUGGUUUGCCACAAAAUCC	574	687	GGAUUUUGUGGCAAACCAU	653
  687	CUCUUUCCAGGAAGGGACU	575	687	CUCUUUCCAGGAAGGGACU	575	705	AGUCCCUUCCUGGAAAGAG	654
  705	UAUAUUGAUCAGUGGAAUA	576	705	UAUAUUGAUCAGUGGAAUA	576	723	UAUUCCACUGAUCAAUAUA	655
  723	AAAGUUAUUGAACAGCUUG	577	723	AAAGUUAUUGAACAGCUUG	577	741	CAAGCUGUUCAAUAACUUU	656
  741	GGAACACCAUGUCCUGAAU	578	741	GGAACACCAUGUCCUGAAU	578	759	AUUCAGGACAUGGUGUUCC	657
  759	UUCAUGAAGAAACUGCAAC	579	759	UUCAUGAAGAAACUGCAAC	579	777	GUUGCAGUUUCUUCAUGAA	658
  777	CCAACAGUAAGGACUUACG	580	777	CCAACAGUAAGGACUUACG	580	795	CGUAAGUCCUUACUGUUGG	659
  795	GUUGAAAACAGACCUAAAU	581	795	GUUGAAAACAGACCUAAAU	581	813	AUUUAGGUCUGUUUUCAAC	660
  813	UAUGCUGGAUAUAGCUUUG	582	813	UAUGCUGGAUAUAGCUUUG	582	831	CAAAGCUAUAUCCAGCAUA	661
  831	GAGAAACUCUUCCCUGAUG	583	831	GAGAAACUCUUCCCUGAUG	583	849	CAUCAGGGAAGAGUUUCUC	662
  849	GUCCUUUUCCCAGCUGACU	584	849	GUCCUUUUCCCAGCUGACU	584	867	AGUCAGCUGGGAAAAGGAC	663
  867	UCAGAACACAACAAACUUA	585	867	UCAGAACACAACAAACUUA	585	885	UAAGUUUGUUGUGUUCUGA	664
  885	AAAGCCAGUCAGGCAAGGG	586	885	AAAGCCAGUCAGGCAAGGG	586	903	CCCUUGCCUGACUGGCUUU	665
  903	GAUUUGUUAUCCAAAAUGC	587	903	GAUUUGUUAUCCAAAAUGC	587	921	GCAUUUUGGAUAACAAAUC	666
  921	CUGGUAAUAGAUGCAUCUA	588	921	CUGGUAAUAGAUGCAUCUA	588	939	UAGAUGCAUCUAUUACCAG	667
  939	AAAAGGAUCUCUGUAGAUG	589	939	AAAAGGAUCUCUGUAGAUG	589	957	CAUCUACAGAGAUCCUUUU	668
  957	GAAGCUCUCCAACACCCGU	590	957	GAAGCUCUCCAACACCCGU	590	975	ACGGGUGUUGGAGAGCUUC	669
  975	UACAUCAAUGUCUGGUAUG	591	975	UACAUCAAUGUCUGGUAUG	591	993	CAUACCAGACAUUGAUGUA	670
  993	GAUCCUUCUGAAGCAGAAG	592	993	GAUCCUUCUGAAGCAGAAG	592	1011	CUUCUGCUUCAGAAGGAUC	671
  1011	GCUCCACCACCAAAGAUCC	593	1011	GCUCCACCACCAAAGAUCC	593	1029	GGAUCUUUGGUGGUGGAGC	672
  1029	CCUGACAAGCAGUUAGAUG	594	1029	CCUGACAAGCAGUUAGAUG	594	1047	CAUCUAACUGCUUGUCAGG	673
  1047	GAAAGGGAACACACAAUAG	595	1047	GAAAGGGAACACACAAUAG	595	1065	CUAUUGUGUGUUCCCUUUC	674
  1065	GAAGAGUGGAAAGAAUUGA	596	1065	GAAGAGUGGAAAGAAUUGA	596	1083	UCAAUUCUUUCCACUCUUC	675
  1083	AUAUAUAAGGAAGUUAUGG	597	1083	AUAUAUAAGGAAGUUAUGG	597	1101	CCAUAACUUCCUUAUAUAU	676
  1101	GACUUGGAGGAGAGAACCA	598	1101	GACUUGGAGGAGAGAACCA	598	1119	UGGUUCUCUCCUCCAAGUC	677
  1119	AAGAAUGGAGUUAUACGGG	599	1119	AAGAAUGGAGUUAUACGGG	599	1137	CCCGUAUAACUCCAUUCUU	678
  1137	GGGCAGCCCUCUCCUUUAG	600	1137	GGGCAGCCCUCUCCUUUAG	600	1155	CUAAAGGAGAGGGCUGCCC	679
  1155	GCACAGGUGCAGCAGUGAU	601	1155	GCACAGGUGCAGCAGUGAU	601	1173	AUCACUGCUGCACCUGUGC	680
  1173	UCAAUGGCUCUCAGCAUCC	602	1173	UCAAUGGCUCUCAGCAUCC	602	1191	GGAUGCUGAGAGCCAUUGA	681
  1191	CAUCAUCAUCGUCGUCUGU	603	1191	CAUCAUCAUCGUCGUCUGU	603	1209	ACAGACGACGAUGAUGAUG	682
  1209	UCAAUGAUGUGUCUUCAAU	604	1209	UCAAUGAUGUGUCUUCAAU	604	1227	AUUGAAGACACAUCAUUGA	683
  1227	UGUCAACAGAUCCGACUUU	605	1227	UGUCAACAGAUCCGACUUU	605	1245	AAAGUCGGAUCUGUUGACA	684
  1245	UGGCCUCUGAUACAGACAG	606	1245	UGGCCUCUGAUACAGACAG	606	1263	CUGUCUGUAUCAGAGGCCA	685
  1263	GCAGUCUAGAAGCAGCAGC	607	1263	GCAGUCUAGAAGCAGCAGC	607	1281	GCUGCUGCUUCUAGACUGC	686
  1281	CUGGGCCUCUGGGCUGCUG	608	1281	CUGGGCCUCUGGGCUGCUG	608	1299	CAGCAGCCCAGAGGCCCAG	687
  1299	GUAGAUGACUACUUGGGCC	609	1299	GUAGAUGACUACUUGGGCC	609	1317	GGCCCAAGUAGUCAUCUAC	688
  1317	CAUCGGGGGGUGGGAGGGA	610	1317	CAUCGGGGGGUGGGAGGGA	610	1335	UCCCUCCCACCCCCCGAUG	689
  1335	AUGGGGAGUCGGUUAGUCA	611	1335	AUGGGGAGUCGGUUAGUCA	611	1353	UGACUAACCGACUCCCCAU	690
  1353	AUUGAUAGAACUACUUUGA	612	1353	AUUGAUAGAACUACUUUGA	612	1371	UCAAAGUAGUUCUAUCAAU	691
  1371	AAAACAAUUCAGUGGUCUU	613	1371	AAAACAAUUCAGUGGUCUU	613	1389	AAGACCACUGAAUUGUUUU	692
  1389	UAUUUUUGGGUGAUUUUUC	614	1389	UAUUUUUGGGUGAUUUUUC	614	1407	GAAAAAUCACCCAAAAAUA	693
  1397	GGUGAUUUUUCAAAAAAUG	615	1397	GGUGAUUUUUCAAAAAAUG	615	1415	CAUUUUUUGAAAAAUCACC	694

  		ID			Seq			Seq
  Pos	Target Sequence	Seq	UPos	Upper seq	ID	LPos	Lower seq	ID

  NM_139012 (MAPK14/p38)

  3	AACCGCGACCACUGGAGCC	695	3	AACCGCGACCACUGGAGCC	695	21	GGCUCCAGUGGUCGCGGUU	904
  21	CUUAGCGGGCGCAGCAGCU	696	21	CUUAGCGGGCGCAGCAGCU	696	39	AGCUGCUGCGCCCGCUAAG	905
  39	UGGAACGGGAGUACUGCGA	697	39	UGGAACGGGAGUACUGCGA	697	57	UCGCAGUACUCCCGUUCCA	906
  57	ACGCAGCCCGGAGUCGGCC	698	57	ACGCAGCCCGGAGUCGGCC	698	75	GGCCGACUCCGGGCUGCGU	907
  75	CUUGUAGGGGCGAAGGUGC	699	75	CUUGUAGGGGCGAAGGUGC	699	93	GCACCUUCGCCCCUACAAG	908
  93	CAGGGAGAUCGCGGCGGGC	700	93	CAGGGAGAUCGCGGCGGGC	700	111	GCCCGCCGCGAUCUCCCUG	909
  111	CGCAGUCUUGAGCGCCGGA	701	111	CGCAGUCUUGAGCGCCGGA	701	129	UCCGGCGCUCAAGACUGCG	910
  129	AGCGCGUCCCUGCCCUUAG	702	129	AGCGCGUCCCUGCCCUUAG	702	147	CUAAGGGCAGGGACGCGCU	911
  147	GCGGGGCUUGCCCCAGUCG	703	147	GCGGGGCUUGCCCCAGUCG	703	165	CGACUGGGGCAAGCCCCGC	912
  165	GCAGGGGCACAUCCAGCCG	704	165	GCAGGGGCACAUCCAGCCG	704	183	CGGCUGGAUGUGCCCCUGC	913
  183	GCUGCGGCUGACAGCAGCC	705	183	GCUGCGGCUGACAGCAGCC	705	201	GGCUGCUGUCAGCCGCAGC	914
  201	CGCGCGCGCGGGAGUCUGC	706	201	CGCGCGCGCGGGAGUCUGC	706	219	GCAGACUCCCGCGCGCGCG	915
  219	CGGGGUCGCGGCAGCCGCA	707	219	CGGGGUCGCGGCAGCCGCA	707	237	UGCGGCUGCCGCGACCCCG	916
  237	ACCUGCGCGGGCGACCAGC	708	237	ACCUGCGCGGGCGACCAGC	708	255	GCUGGUCGCCCGCGCAGGU	917
  255	CGCAAGGUCCCCGCCCGGC	709	255	CGCAAGGUCCCCGCCCGGC	709	273	GCCGGGCGGGGACCUUGCG	918
  273	CUGGGCGGGCAGCAAGGGC	710	273	CUGGGCGGGCAGCAAGGGC	710	291	GCCCUUGCUGCCCGCCCAG	919
  291	CCGGGGAGAGGGUGCGGGU	711	291	CCGGGGAGAGGGUGCGGGU	711	309	ACCCGCACCCUCUCCCCGG	920
  309	UGCAGGCGGGGGCCCCACA	712	309	UGCAGGCGGGGGCCCCACA	712	327	UGUGGGGCCCCCGCCUGCA	921
  327	AGGGCCACCUUCUUGCCCG	713	327	AGGGCCACCUUCUUGCCCG	713	345	CGGGCAAGAAGGUGGCCCU	922
  345	GGCGGCUGCCGCUGGAAAA	714	345	GGCGGCUGCCGCUGGAAAA	714	363	UUUUCCAGCGGCAGCCGCC	923
  363	AUGUCUCAGGAGAGGCCCA	715	363	AUGUCUCAGGAGAGGCCCA	715	381	UGGGCCUCUCCUGAGACAU	924
  381	ACGUUCUACCGGCAGGAGC	716	381	ACGUUCUACCGGCAGGAGC	716	399	GCUCCUGCCGGUAGAACGU	925
  399	CUGAACAAGACAAUCUGGG	717	399	CUGAACAAGACAAUCUGGG	717	417	CCCAGAUUGUCUUGUUCAG	926
  417	GAGGUGCCCGAGCGUUACC	718	417	GAGGUGCCCGAGCGUUACC	718	435	GGUAACGCUCGGGCACCUC	927
  435	CAGAACCUGUCUCCAGUGG	719	435	CAGAACCUGUCUCCAGUGG	719	453	CCACUGGAGACAGGUUCUG	928
  453	GGCUCUGGCGCCUAUGGCU	720	453	GGCUCUGGCGCCUAUGGCU	720	471	AGCCAUAGGCGCCAGAGCC	929
  471	UCUGUGUGUGCUGCUUUUG	721	471	UCUGUGUGUGCUGCUUUUG	721	489	CAAAAGCAGCACACACAGA	930
  489	GACACAAAAACGGGGUUAC	722	489	GACACAAAAACGGGGUUAC	722	507	GUAACCCCGUUUUUGUGUC	931
  507	CGUGUGGCAGUGAAGAAGC	723	507	CGUGUGGCAGUGAAGAAGC	723	525	GCUUCUUCACUGCCACACG	932
  525	CUCUCCAGACCAUUUCAGU	724	525	CUCUCCAGACCAUUUCAGU	724	543	ACUGAAAUGGUCUGGAGAG	933
  543	UCCAUCAUUCAUGCGAAAA	725	543	UCCAUCAUUCAUGCGAAAA	725	561	UUUUCGCAUGAAUGAUGGA	934
  561	AGAACCUACAGAGAACUGC	726	561	AGAACCUACAGAGAACUGC	726	579	GCAGUUCUCUGUAGGUUCU	935
  579	CGGUUACUUAAACAUAUGA	727	579	CGGUUACUUAAACAUAUGA	727	597	UCAUAUGUUUAAGUAACCG	936
  597	AAACAUGAAAAUGUGAUUG	728	597	AAACAUGAAAAUGUGAUUG	728	615	CAAUCACAUUUUCAUGUUU	937
  615	GGUCUGUUGGACGUUUUUA	729	615	GGUCUGUUGGACGUUUUUA	729	633	UAAAAACGUCCAACAGACC	938
  633	ACACCUGCAAGGUCUCUGG	730	633	ACACCUGCAAGGUCUCUGG	730	651	CCAGAGACCUUGCAGGUGU	939
  651	GAGGAAUUCAAUGAUGUGU	731	651	GAGGAAUUCAAUGAUGUGU	731	669	ACACAUCAUUGAAUUCCUC	940
  669	UAUCUGGUGACCCAUCUCA	732	669	UAUCUGGUGACCCAUCUCA	732	687	UGAGAUGGGUCACCAGAUA	941
  687	AUGGGGGCAGAUCUGAACA	733	687	AUGGGGGCAGAUCUGAACA	733	705	UGUUCAGAUCUGCCCCCAU	942
  705	AACAUUGUGAAAUGUCAGA	734	705	AACAUUGUGAAAUGUCAGA	734	723	UCUGACAUUUCACAAUGUU	943
  723	AAGCUUACAGAUGACCAUG	735	723	AAGCUUACAGAUGACCAUG	735	741	CAUGGUCAUCUGUAAGCUU	944
  741	GUUCAGUUCCUUAUCUACC	736	741	GUUCAGUUCCUUAUCUACC	736	759	GGUAGAUAAGGAACUGAAC	945
  759	CAAAUUCUCCGAGGUCUAA	737	759	CAAAUUCUCCGAGGUCUAA	737	777	UUAGACCUCGGAGAAUUUG	946
  777	AAGUAUAUACAUUCAGCUG	738	777	AAGUAUAUACAUUCAGCUG	738	795	CAGCUGAAUGUAUAUACUU	947
  795	GACAUAAUUCACAGGGACC	739	795	GACAUAAUUCACAGGGACC	739	813	GGUCCCUGUGAAUUAUGUC	948
  813	CUAAAACCUAGUAAUCUAG	740	813	CUAAAACCUAGUAAUCUAG	740	831	CUAGAUUACUAGGUUUUAG	949
  831	GCUGUGAAUGAAGACUGUG	741	831	GCUGUGAAUGAAGACUGUG	741	849	CACAGUCUUCAUUCACAGC	950
  849	GAGCUGAAGAUUCUGGAUU	742	849	GAGCUGAAGAUUCUGGAUU	742	867	AAUCCAGAAUCUUCAGCUC	951
  867	UUUGGACUGGCUCGGCACA	743	867	UUUGGACUGGCUCGGCACA	743	885	UGUGCCGAGCCAGUCCAAA	952
  885	ACAGAUGAUGAAAUGACAG	744	885	ACAGAUGAUGAAAUGACAG	744	903	CUGUCAUUUCAUCAUCUGU	953
  903	GGCUACGUGGCCACUAGGU	745	903	GGCUACGUGGCCACUAGGU	745	921	ACCUAGUGGCCACGUAGCC	954
  921	UGGUACAGGGCUCCUGAGA	746	921	UGGUACAGGGCUCCUGAGA	746	939	UCUCAGGAGCCCUGUACCA	955
  939	AUCAUGCUGAACUGGAUGC	747	939	AUCAUGCUGAACUGGAUGC	747	957	GCAUCCAGUUCAGCAUGAU	956
  957	CAUUACAACCAGACAGUUG	748	957	CAUUACAACCAGACAGUUG	748	975	CAACUGUCUGGUUGUAAUG	957
  975	GAUAUUUGGUCAGUGGGAU	749	975	GAUAUUUGGUCAGUGGGAU	749	993	AUCCCACUGACCAAAUAUC	958
  993	UGCAUAAUGGCCGAGCUGU	750	993	UGCAUAAUGGCCGAGCUGU	750	1011	ACAGCUCGGCCAUUAUGCA	959
  1011	UUGACUGGAAGAACAUUGU	751	1011	UUGACUGGAAGAACAUUGU	751	1029	ACAAUGUUCUUCCAGUCAA	960
  1029	UUUCCUGGUACAGACCAUA	752	1029	UUUCCUGGUACAGACCAUA	752	1047	UAUGGUCUGUACCAGGAAA	961
  1047	AUUGAUCAGUUGAAGCUCA	753	1047	AUUGAUCAGUUGAAGCUCA	753	1065	UGAGCUUCAACUGAUCAAU	962
  1065	AUUUUAAGACUCGUUGGAA	754	1065	AUUUUAAGACUCGUUGGAA	754	1083	UUCCAACGAGUCUUAAAAU	963
  1083	ACCCCAGGGGCUGAGCUUU	755	1083	ACCCCAGGGGCUGAGCUUU	755	1101	AAAGCUCAGCCCCUGGGGU	964
  1101	UUGAAGAAAAUCUCCUCAG	756	1101	UUGAAGAAAAUCUCCUCAG	756	1119	CUGAGGAGAUUUUCUUCAA	965
  1119	GAGUCUGCAAGAAACUAUA	757	1119	GAGUCUGCAAGAAACUAUA	757	1137	UAUAGUUUCUUGCAGACUC	966
  1137	AUUCAGUCUUUGACUCAGA	758	1137	AUUCAGUCUUUGACUCAGA	758	1155	UCUGAGUCAAAGACUGAAU	967
  1155	AUGCCGAAGAUGAACUUUG	759	1155	AUGCCGAAGAUGAACUUUG	759	1173	CAAAGUUCAUCUUCGGCAU	968
  1173	GCGAAUGUAUUUAUUGGUG	760	1173	GCGAAUGUAUUUAUUGGUG	760	1191	CACCAAUAAAUACAUUCGC	969
  1191	GCCAAUCCCCUGGCUGUCG	761	1191	GCCAAUCCCCUGGCUGUCG	761	1209	CGACAGCCAGGGGAUUGGC	970
  1209	GACUUGCUGGAGAAGAUGC	762	1209	GACUUGCUGGAGAAGAUGC	762	1227	GCAUCUUCUCCAGCAAGUC	971
  1227	CUUGUAUUGGACUCAGAUA	763	1227	CUUGUAUUGGACUCAGAUA	763	1245	UAUCUGAGUCCAAUACAAG	972
  1245	AAGAGAAUUACAGCGGCCC	764	1245	AAGAGAAUUACAGCGGCCC	764	1263	GGGCCGCUGUAAUUCUCUU	973
  1263	CAAGCCCUUGCACAUGCCU	765	1263	CAAGCCCUUGCACAUGCCU	765	1281	AGGCAUGUGCAAGGGCUUG	974
  1281	UACUUUGCUCAGUACCACG	766	1281	UACUUUGCUCAGUACCACG	766	1299	CGUGGUACUGAGCAAAGUA	975
  1299	GAUCCUGAUGAUGAACCAG	767	1299	GAUCCUGAUGAUGAACCAG	767	1317	CUGGUUCAUCAUCAGGAUC	976
  1317	GUGGCCGAUCCUUAUGAUC	768	1317	GUGGCCGAUCCUUAUGAUC	768	1335	GAUCAUAAGGAUCGGCCAC	977
  1335	CAGUCCUUUGAAAGCAGGG	769	1335	CAGUCCUUUGAAAGCAGGG	769	1353	CCCUGCUUUCAAAGGACUG	978
  1353	GACCUCCUUAUAGAUGAGU	770	1353	GACCUCCUUAUAGAUGAGU	770	1371	ACUCAUCUAUAAGGAGGUC	979
  1371	UGGAAAAGCCUGACCUAUG	771	1371	UGGAAAAGCCUGACCUAUG	771	1389	CAUAGGUCAGGCUUUUCCA	980
  1389	GAUGAAGUCAUCAGCUUUG	772	1389	GAUGAAGUCAUCAGCUUUG	772	1407	CAAAGCUGAUGACUUCAUC	981
  1407	GUGCCACCACCCCUUGACC	773	1407	GUGCCACCACCCCUUGACC	773	1425	GGUCAAGGGGUGGUGGCAC	982
  1425	CAAGAAGAGAUGGAGUCCU	774	1425	CAAGAAGAGAUGGAGUCCU	774	1443	AGGACUCCAUCUCUUCUUG	983
  1443	UGAGCACCUGGUUUCUGUU	775	1443	UGAGCACCUGGUUUCUGUU	775	1461	AACAGAAACCAGGUGCUCA	984
  1461	UCUGUUGAUCCCACUUCAC	776	1461	UCUGUUGAUCCCACUUCAC	776	1479	GUGAAGUGGGAUCAACAGA	985
  1479	CUGUGAGGGGAAGGCCUUU	777	1479	CUGUGAGGGGAAGGCCUUU	777	1497	AAAGGCCUUCCCCUCACAG	986
  1497	UUCACGGGAACUCUCCAAA	778	1497	UUCACGGGAACUCUCCAAA	778	1515	UUUGGAGAGUUCCCGUGAA	987
  1515	AUAUUAUUCAAGUGCCUCU	779	1515	AUAUUAUUCAAGUGCCUCU	779	1533	AGAGGCACUUGAAUAAUAU	988
  1533	UUGUUGCAGAGAUUUCCUC	780	1533	UUGUUGCAGAGAUUUCCUC	780	1551	GAGGAAAUCUCUGCAACAA	989
  1551	CCAUGGUGGAAGGGGGUGU	781	1551	CCAUGGUGGAAGGGGGUGU	781	1569	ACACCCCCUUCCACCAUGG	990
  1569	UGCGUGCGUGUGCGUGCGU	782	1569	UGCGUGCGUGUGCGUGCGU	782	1587	ACGCACGCACACGCACGCA	991
  1587	UGUUAGUGUGUGUGCAUGU	783	1587	UGUUAGUGUGUGUGCAUGU	783	1605	ACAUGCACACACACUAACA	992
  1605	UGUGUGUCUGUCUUUGUGG	784	1605	UGUGUGUCUGUCUUUGUGG	784	1623	CCACAAAGACAGACACACA	993
  1623	GGAGGGUAAGACAAUAUGA	785	1623	GGAGGGUAAGACAAUAUGA	785	1641	UCAUAUUGUCUUACCCUCC	994
  1641	AACAAACUAUGAUCACAGU	786	1641	AACAAACUAUGAUCACAGU	786	1659	ACUGUGAUCAUAGUUUGUU	995
  1659	UGACUUUACAGGAGGUUGU	787	1659	UGACUUUACAGGAGGUUGU	787	1677	ACAACCUCCUGUAAAGUCA	996
  1677	UGGAUGCUCCAGGGCAGCC	788	1677	UGGAUGCUCCAGGGCAGCC	788	1695	GGCUGCCCUGGAGCAUCCA	997
  1695	CUCCACCUUGCUCUUCUUU	789	1695	CUCCACCUUGCUCUUCUUU	789	1713	AAAGAAGAGCAAGGUGGAG	998
  1713	UCUGAGAGUUGGCUCAGGC	790	1713	UCUGAGAGUUGGCUCAGGC	790	1731	GCCUGAGCCAACUCUCAGA	999
  1731	CAGACAAGAGCUGCUGUCC	791	1731	CAGACAAGAGCUGCUGUCC	791	1749	GGACAGCAGCUCUUGUCUG	1000
  1749	CUUUUAGGAAUAUGUUCAA	792	1749	CUUUUAGGAAUAUGUUCAA	792	1767	UUGAACAUAUUCCUAAAAG	1001
  1767	AUGCAAAGUAAAAAAAUAU	793	1767	AUGCAAAGUAAAAAAAUAU	793	1785	AUAUUUUUUUACUUUGCAU	1002
  1785	UGAAUUGUCCCCAAUCCCG	794	1785	UGAAUUGUCCCCAAUCCCG	794	1803	CGGGAUUGGGGACAAUUCA	1003
  1803	GGUCAUGCUUUUGCCACUU	795	1803	GGUCAUGCUUUUGCCACUU	795	1821	AAGUGGCAAAAGCAUGACC	1004
  1821	UUGGCUUCUCCUGUGACCC	796	1821	UUGGCUUCUCCUGUGACCC	796	1839	GGGUCACAGGAGAAGCCAA	1005
  1839	CCACCUUGACGGUGGGGCG	797	1839	CCACCUUGACGGUGGGGCG	797	1857	CGCCCCACCGUCAAGGUGG	1006
  1857	GUAGACUUGACAACAUCCC	798	1857	GUAGACUUGACAACAUCCC	798	1875	GGGAUGUUGUCAAGUCUAC	1007
  1875	CACAGUGGCACGGAGAGAA	799	1875	CACAGUGGCACGGAGAGAA	799	1893	UUCUCUCCGUGCCACUGUG	1008
  1893	AGGCCCAUACCUUCUGGUU	800	1893	AGGCCCAUACCUUCUGGUU	800	1911	AACCAGAAGGUAUGGGCCU	1009
  1911	UGCUUCAGACCUGACACCG	801	1911	UGCUUCAGACCUGACACCG	801	1929	CGGUGUCAGGUCUGAAGCA	1010
  1929	GUCCCUCAGUGAUACGUAC	802	1929	GUCCCUCAGUGAUACGUAC	802	1947	GUACGUAUCACUGAGGGAC	1011
  1947	CAGCCAAAAAGGACCAACU	803	1947	CAGCCAAAAAGGACCAACU	803	1965	AGUUGGUCCUUUUUGGCUG	1012
  1965	UGGCUUCUGUGCACUAGCC	804	1965	UGGCUUCUGUGCACUAGCC	804	1983	GGCUAGUGCACAGAAGCCA	1013
  1983	CUGUGAUUAACUUGCUUAG	805	1983	CUGUGAUUAACUUGCUUAG	805	2001	CUAAGCAAGUUAAUCACAG	1014
  2001	GUAUGGUUCUCAGAUCUUG	806	2001	GUAUGGUUCUCAGAUCUUG	806	2019	CAAGAUCUGAGAACCAUAC	1015
  2019	GACAGUAUAUUUGAAACUG	807	2019	GACAGUAUAUUUGAAACUG	807	2037	CAGUUUCAAAUAUACUGUC	1016
  2037	GUAAAUAUGUUUGUGCCUU	808	2037	GUAAAUAUGUUUGUGCCUU	808	2055	AAGGCACAAACAUAUUUAC	1017
  2055	UAAAAGGAGAGAAGAAAGU	809	2055	UAAAAGGAGAGAAGAAAGU	809	2073	ACUUUCUUCUCUCCUUUUA	1018
  2073	UGUAGAUAGUUAAAAGACU	810	2073	UGUAGAUAGUUAAAAGACU	810	2091	AGUCUUUUAACUAUCUACA	1019
  2091	UGCAGCUGCUGAAGUUCUG	811	2091	UGCAGCUGCUGAAGUUCUG	811	2109	CAGAACUUCAGCAGCUGCA	1020
  2109	GAGCCGGGCAAGUCGAGAG	812	2109	GAGCCGGGCAAGUCGAGAG	812	2127	CUCUCGACUUGCCCGGCUC	1021
  2127	GGGCUGUUGGACAGCUGCU	813	2127	GGGCUGUUGGACAGCUGCU	813	2145	AGCAGCUGUCCAACAGCCC	1022
  2145	UUGUGGGCCCGGAGUAAUC	814	2145	UUGUGGGCCCGGAGUAAUC	814	2163	GAUUACUCCGGGCCCACAA	1023
  2163	CAGGCAGCCUUCAUAGGCG	815	2163	CAGGCAGCCUUCAUAGGCG	815	2181	CGCCUAUGAAGGCUGCCUG	1024
  2181	GGUCAUGUGUGCAUGUGAG	816	2181	GGUCAUGUGUGCAUGUGAG	816	2199	CUCACAUGCACACAUGACC	1025
  2199	GCACAUGCGUAUAUGUGCG	817	2199	GCACAUGCGUAUAUGUGCG	817	2217	CGCACAUAUACGCAUGUGC	1026
  2217	GUCUCUCUUUCUCCCUCAC	818	2217	GUCUCUCUUUCUCCCUCAC	818	2235	GUGAGGGAGAAAGAGAGAC	1027
  2235	CCCCCAGGUGUUGCCAUUU	819	2235	CCCCCAGGUGUUGCCAUUU	819	2253	AAAUGGCAACACCUGGGGG	1028
  2253	UCUCUGCUUACCCUUCACC	820	2253	UCUCUGCUUACCCUUCACC	820	2271	GGUGAAGGGUAAGCAGAGA	1029
  2271	CUUUGGUGCAGAGGUUUCU	821	2271	CUUUGGUGCAGAGGUUUCU	821	2289	AGAAACCUCUGCACCAAAG	1030
  2289	UUGAAUAUCUGCCCCAGUA	822	2289	UUGAAUAUCUGCCCCAGUA	822	2307	UACUGGGGCAGAUAUUCAA	1031
  2307	AGUCAGAAGCAGGUUCUUG	823	2307	AGUCAGAAGCAGGUUCUUG	823	2325	CAAGAACCUGCUUCUGACU	1032
  2325	GAUGUCAUGUACUUCCUGU	824	2325	GAUGUCAUGUACUUCCUGU	824	2343	ACAGGAAGUACAUGACAUC	1033
  2343	UGUACUCUUUAUUUCUAGC	825	2343	UGUACUCUUUAUUUCUAGC	825	2361	GCUAGAAAUAAAGAGUACA	1034
  2361	CAGAGUGAGGAUGUGUUUU	826	2361	CAGAGUGAGGAUGUGUUUU	826	2379	AAAACACAUCCUCACUCUG	1035
  2379	UGCACGUCUUGCUAUUUGA	827	2379	UGCACGUCUUGCUAUUUGA	827	2397	UCAAAUAGCAAGACGUGCA	1036
  2397	AGCAUGCACAGCUGCUUGU	828	2397	AGCAUGCACAGCUGCUUGU	828	2415	ACAAGCAGCUGUGCAUGCU	1037
  2415	UCCUGCUCUCUUCAGGAGG	829	2415	UCCUGCUCUCUUCAGGAGG	829	2433	CCUCCUGAAGAGAGCAGGA	1038
  2433	GCCCUGGUGUCAGGCAGGU	830	2433	GCCCUGGUGUCAGGCAGGU	830	2451	ACCUGCCUGACACCAGGGC	1039
  2451	UUUGCCAGUGAAGACUUCU	831	2451	UUUGCCAGUGAAGACUUCU	831	2469	AGAAGUCUUCACUGGCAAA	1040
  2469	UUGGGUAGUUUAGAUCCCA	832	2469	UUGGGUAGUUUAGAUCCCA	832	2487	UGGGAUCUAAACUACCCAA	1041
  2487	AUGUCACCUCAGCUGAUAU	833	2487	AUGUCACCUCAGCUGAUAU	833	2505	AUAUCAGCUGAGGUGACAU	1042
  2505	UUAUGGCAAGUGAUAUCAC	834	2505	UUAUGGCAAGUGAUAUCAC	834	2523	GUGAUAUCACUUGCCAUAA	1043
  2523	CCUCUCUUCAGCCCCUAGU	835	2523	CCUCUCUUCAGCCCCUAGU	835	2541	ACUAGGGGCUGAAGAGAGG	1044
  2541	UGCUAUUCUGUGUUGAACA	836	2541	UGCUAUUCUGUGUUGAACA	836	2559	UGUUCAACACAGAAUAGCA	1045
  2559	ACAAUUGAUACUUCAGGUG	837	2559	ACAAUUGAUACUUCAGGUG	837	2577	CACCUGAAGUAUCAAUUGU	1046
  2577	GCUUUUGAUGUGAAAAUCA	838	2577	GCUUUUGAUGUGAAAAUCA	838	2595	UGAUUUUCACAUCAAAAGC	1047
  2595	AUGAAAAGAGGAACAGGUG	839	2595	AUGAAAAGAGGAACAGGUG	839	2613	CACCUGUUCCUCUUUUCAU	1048
  2613	GGAUGUAUAGCAUUUUUAU	840	2613	GGAUGUAUAGCAUUUUUAU	840	2631	AUAAAAAUGCUAUACAUCC	1049
  2631	UUCAUGCCAUCUGUUUUCA	841	2631	UUCAUGCCAUCUGUUUUCA	841	2649	UGAAAACAGAUGGCAUGAA	1050
  2649	AACCAACUAUUUUUGAGGA	842	2649	AACCAACUAUUUUUGAGGA	842	2667	UCCUCAAAAAUAGUUGGUU	1051
  2667	AAUUAUCAUGGGAAAAGAC	843	2667	AAUUAUCAUGGGAAAAGAC	843	2685	GUCUUUUCCCAUGAUAAUU	1052
  2685	CCAGGGCUUUUCCCAGGAA	844	2685	CCAGGGCUUUUCCCAGGAA	844	2703	UUCCUGGGAAAAGCCCUGG	1053
  2703	AUAUCCCAAACUUCGGAAA	845	2703	AUAUCCCAAACUUCGGAAA	845	2721	UUUCCGAAGUUUGGGAUAU	1054
  2721	ACAAGUUAUUCUCUUCACU	846	2721	ACAAGUUAUUCUCUUCACU	846	2739	AGUGAAGAGAAUAACUUGU	1055
  2739	UCCCAAUAACUAAUGCUAA	847	2739	UCCCAAUAACUAAUGCUAA	847	2757	UUAGCAUUAGUUAUUGGGA	1056
  2757	AGAAAUGCUGAAAAUCAAA	848	2757	AGAAAUGCUGAAAAUCAAA	848	2775	UUUGAUUUUCAGCAUUUCU	1057
  2775	AGUAAAAAAUUAAAGCCCA	849	2775	AGUAAAAAAUUAAAGCCCA	849	2793	UGGGCUUUAAUUUUUUACU	1058
  2793	AUAAGGCCAGAAACUCCUU	850	2793	AUAAGGCCAGAAACUCCUU	850	2811	AAGGAGUUUCUGGCCUUAU	1059
  2811	UUUGCUGUCUUUCUCUAAA	851	2811	UUUGCUGUCUUUCUCUAAA	851	2829	UUUAGAGAAAGACAGCAAA	1060
  2829	AUAUGAUUACUUUAAAAUA	852	2829	AUAUGAUUACUUUAAAAUA	852	2847	UAUUUUAAAGUAAUCAUAU	1061
  2847	AAAAAAGUAACAAGGUGUC	853	2847	AAAAAAGUAACAAGGUGUC	853	2865	GACACCUUGUUACUUUUUU	1062
  2865	CUUUUCCACUCCUAUGGAA	854	2865	CUUUUCCACUCCUAUGGAA	854	2883	UUCCAUAGGAGUGGAAAAG	1063
  2883	AAAGGGUCUUCUUGGCAGC	855	2883	AAAGGGUCUUCUUGGCAGC	855	2901	GCUGCCAAGAAGACCCUUU	1064
  2901	CUUAACAUUGACUUCUUGG	856	2901	CUUAACAUUGACUUCUUGG	856	2919	CCAAGAAGUCAAUGUUAAG	1065
  2919	GUUUGGGGAGAAAUAAAUU	857	2919	GUUUGGGGAGAAAUAAAUU	857	2937	AAUUUAUUUCUCCCCAAAC	1066
  2937	UUUGUUUCAGAAUUUUGUA	858	2937	UUUGUUUCAGAAUUUUGUA	858	2955	UACAAAAUUCUGAAACAAA	1067
  2955	AUAUUGUAGGAAUCCCUUU	859	2955	AUAUUGUAGGAAUCCCUUU	859	2973	AAAGGGAUUCCUACAAUAU	1068
  2973	UGAGAAUGUGAUUCCUUUU	860	2973	UGAGAAUGUGAUUCCUUUU	860	2991	AAAAGGAAUCACAUUCUCA	1069
  2991	UGAUGGGGAGAAAGGGCAA	861	2991	UGAUGGGGAGAAAGGGCAA	861	3009	UUGCCCUUUCUCCCCAUCA	1070
  3009	AAUUAUUUUAAUAUUUUGU	862	3009	AAUUAUUUUAAUAUUUUGU	862	3027	ACAAAAUAUUAAAAUAAUU	1071
  3027	UAUUUUCAACUUUAUAAAG	863	3027	UAUUUUCAACUUUAUAAAG	863	3045	CUUUAUAAAGUUGAAAAUA	1072
  3045	GAUAAAAUAUCCUCAGGGG	864	3045	GAUAAAAUAUCCUCAGGGG	864	3063	CCCCUGAGGAUAUUUUAUC	1073
  3063	GUGGAGAAGUGUCGUUUUC	865	3063	GUGGAGAAGUGUCGUUUUC	865	3081	GAAAACGACACUUCUCCAC	1074
  3081	CAUAACUUGCUGAAUUUCA	866	3081	CAUAACUUGCUGAAUUUCA	866	3099	UGAAAUUCAGCAAGUUAUG	1075
  3099	AGGCAUUUUGUUCUACAUG	867	3099	AGGCAUUUUGUUCUACAUG	867	3117	CAUGUAGAACAAAAUGCCU	1076
  3117	GAGGACUCAUAUAUUUAAG	868	3117	GAGGACUCAUAUAUUUAAG	868	3135	CUUAAAUAUAUGAGUCCUC	1077
  3135	GCCUUUUGUGUAAUAAGAA	869	3135	GCCUUUUGUGUAAUAAGAA	869	3153	UUCUUAUUACACAAAAGGC	1078
  3153	AAGUAUAAAGUCACUUCCA	870	3153	AAGUAUAAAGUCACUUCCA	870	3171	UGGAAGUGACUUUAUACUU	1079
  3171	AGUGUUGGCUGUGUGACAG	871	3171	AGUGUUGGCUGUGUGACAG	871	3189	CUGUCACACAGCCAACACU	1080
  3189	GAAUCUUGUAUUUGGGCCA	872	3189	GAAUCUUGUAUUUGGGCCA	872	3207	UGGCCCAAAUACAAGAUUC	1081
  3207	AAGGUGUUUCCAUUUCUCA	873	3207	AAGGUGUUUCCAUUUCUCA	873	3225	UGAGAAAUGGAAACACCUU	1082
  3225	AAUCAGUGCAGUGAUACAU	874	3225	AAUCAGUGCAGUGAUACAU	874	3243	AUGUAUCACUGCACUGAUU	1083
  3243	UGUACUCCAGAGGGACAGG	875	3243	UGUACUCCAGAGGGACAGG	875	3261	CCUGUCCCUCUGGAGUACA	1084
  3261	GGUGGACCCCCUGAGUCAA	876	3261	GGUGGACCCCCUGAGUCAA	876	3279	UUGACUCAGGGGGUCCACC	1085
  3279	ACUGGAGCAAGAAGGAAGG	877	3279	ACUGGAGCAAGAAGGAAGG	877	3297	CCUUCCUUCUUGCUCCAGU	1086
  3297	GAGGCAGACUGAUGGCGAU	878	3297	GAGGCAGACUGAUGGCGAU	878	3315	AUCGCCAUCAGUCUGCCUC	1087
  3315	UUCCCUCUCACCCGGGACU	879	3315	UUCCCUCUCACCCGGGACU	879	3333	AGUCCCGGGUGAGAGGGAA	1088
  3333	UCUCCCCCUUUCAAGGAAA	880	3333	UCUCCCCCUUUCAAGGAAA	880	3351	UUUCCUUGAAAGGGGGAGA	1089
  3351	AGUGAACCUUUAAAGUAAA	881	3351	AGUGAACCUUUAAAGUAAA	881	3369	UUUACUUUAAAGGUUCACU	1090
  3369	AGGCCUCAUCUCCUUUAUU	882	3369	AGGCCUCAUCUCCUUUAUU	882	3387	AAUAAAGGAGAUGAGGCCU	1091
  3387	UGCAGUUCAAAUCCUCACC	883	3387	UGCAGUUCAAAUCCUCACC	883	3405	GGUGAGGAUUUGAACUGCA	1092
  3405	CAUCCACAGCAAGAUGAAU	884	3405	CAUCCACAGCAAGAUGAAU	884	3423	AUUCAUCUUGCUGUGGAUG	1093
  3423	UUUUAUCAGCCAUGUUUGG	885	3423	UUUUAUCAGCCAUGUUUGG	885	3441	CCAAACAUGGCUGAUAAAA	1094
  3441	GUUGUAAAUGCUCGUGUGA	886	3441	GUUGUAAAUGCUCGUGUGA	886	3459	UCACACGAGCAUUUACAAC	1095
  3459	AUUUCCUACAGAAAUACUG	887	3459	AUUUCCUACAGAAAUACUG	887	3477	CAGUAUUUCUGUAGGAAAU	1096
  3477	GCUCUGAAUAUUUUGUAAU	888	3477	GCUCUGAAUAUUUUGUAAU	888	3495	AUUACAAAAUAUUCAGAGC	1097
  3495	UAAAGGUCUUUGCACAUGU	889	3495	UAAAGGUCUUUGCACAUGU	889	3513	ACAUGUGCAAAGACCUUUA	1098
  3513	UGACCACAUACGUGUUAGG	890	3513	UGACCACAUACGUGUUAGG	890	3531	CCUAACACGUAUGUGGUCA	1099
  3531	GAGGCUGCAUGCUCUGGAA	891	3531	GAGGCUGCAUGCUCUGGAA	891	3549	UUCCAGAGCAUGCAGCCUC	1100
  3549	AGCCUGGACUCUAAGCUGG	892	3549	AGCCUGGACUCUAAGCUGG	892	3567	CCAGCUUAGAGUCCAGGCU	1101
  3567	GAGCUCUUGGAAGAGCUCU	893	3567	GAGCUCUUGGAAGAGCUCU	893	3585	AGAGCUCUUCCAAGAGCUC	1102
  3585	UUCGGUUUCUGAGCAUAAU	894	3585	UUCGGUUUCUGAGCAUAAU	894	3603	AUUAUGCUCAGAAACCGAA	1103
  3603	UGCUCCCAUCUCCUGAUUU	895	3603	UGCUCCCAUCUCCUGAUUU	895	3621	AAAUCAGGAGAUGGGAGCA	1104
  3621	UCUCUGAACAGAAAACAAA	896	3621	UCUCUGAACAGAAAACAAA	896	3639	UUUGUUUUCUGUUCAGAGA	1105
  3639	AAGAGAGAAUGAGGGAAAU	897	3639	AAGAGAGAAUGAGGGAAAU	897	3657	AUUUCCCUCAUUCUCUCUU	1106
  3657	UUGCUAUUUUAUUUGUAUU	898	3657	UUGCUAUUUUAUUUGUAUU	898	3675	AAUACAAAUAAAAUAGCAA	1107
  3675	UCAUGAACUUGGCUGUAAU	899	3675	UCAUGAACUUGGCUGUAAU	899	3693	AUUACAGCCAAGUUCAUGA	1108
  3693	UCAGUUAUGCCGUAUAGGA	900	3693	UCAGUUAUGCCGUAUAGGA	900	3711	UCCUAUACGGCAUAACUGA	1109
  3711	AUGUCAGACAAUACCACUG	901	3711	AUGUCAGACAAUACCACUG	901	3729	CAGUGGUAUUGUCUGACAU	1110
  3729	GGUUAAAAUAAAGCCUAUU	902	3729	GGUUAAAAUAAAGCCUAUU	902	3747	AAUAGGCUUUAUUUUAACC	1111
  3737	UAAAGCCUAUUUUUCAAAU	903	3737	UAAAGCCUAUUUUUCAAAU	903	3755	AUUUGAAAAAUAGGCUUUA	1112

  		Seq			Seq			Seq
  Pos	Seq	ID	UPos	Upper seq	ID	LPos	Lower seq	ID

  hJUN NM_002228

  3	AGUUGCACUGAGUGUGGCU	1247	3	AGUUGCACUGAGUGUGGCU	1247	21	AGCCACACUCAGUGCAACU	1428
  21	UGAAGCAGCGAGGCGGGAG	1248	21	UGAAGCAGCGAGGCGGGAG	1248	39	CUCCCGCCUCGCUGCUUCA	1429
  39	GUGGAGGUGCGCGGAGUCA	1249	39	GUGGAGGUGCGCGGAGUCA	1249	57	UGACUCCGCGCACCUCCAC	1430
  57	AGGCAGACAGACAGACACA	1250	57	AGGCAGACAGACAGACACA	1250	75	UGUGUCUGUCUGUCUGCCU	1431
  75	AGCCAGCCAGCCAGGUCGG	1251	75	AGCCAGCCAGCCAGGUCGG	1251	93	CCGACCUGGCUGGCUGGCU	1432
  93	GCAGUAUAGUCCGAACUGC	1252	93	GCAGUAUAGUCCGAACUGC	1252	111	GCAGUUCGGACUAUACUGC	1433
  111	CAAAUCUUAUUUUCUUUUC	1253	111	CAAAUCUUAUUUUCUUUUC	1253	129	GAAAAGAAAAUAAGAUUUG	1434
  129	CACCUUCUCUCUAACUGCC	1254	129	CACCUUCUCUCUAACUGCC	1254	147	GGCAGUUAGAGAGAAGGUG	1435
  147	CCAGAGCUAGCGCCUGUGG	1255	147	CCAGAGCUAGCGCCUGUGG	1255	165	CCACAGGCGCUAGCUCUGG	1436
  165	GCUCCCGGGCUGGUGGUUC	1256	165	GCUCCCGGGCUGGUGGUUC	1256	183	GAACCACCAGCCCGGGAGC	1437
  183	CGGGAGUGUCCAGAGAGCC	1257	183	CGGGAGUGUCCAGAGAGCC	1257	201	GGCUCUCUGGACACUCCCG	1438
  201	CUUGUCUCCAGCCGGCCCC	1258	201	CUUGUCUCCAGCCGGCCCC	1258	219	GGGGCCGGCUGGAGACAAG	1439
  219	CGGGAGGAGAGCCCUGCUG	1259	219	CGGGAGGAGAGCCCUGCUG	1259	237	CAGCAGGGCUCUCCUCCCG	1440
  237	GCCCAGGCGCUGUUGACAG	1260	237	GCCCAGGCGCUGUUGACAG	1260	255	CUGUCAACAGCGCCUGGGC	1441
  255	GCGGCGGAAAGCAGCGGUA	1261	255	GCGGCGGAAAGCAGCGGUA	1261	273	UACCGCUGCUUUCCGCCGC	1442
  273	ACCCCACGCGCCCGCCGGG	1262	273	ACCCCACGCGCCCGCCGGG	1262	291	CCCGGCGGGCGCGUGGGGU	1443
  291	GGGACGUCGGCGAGCGGCU	1263	291	GGGACGUCGGCGAGCGGCU	1263	309	AGCCGCUCGCCGACGUCCC	1444
  309	UGCAGCAGCAAAGAACUUU	1264	309	UGCAGCAGCAAAGAACUUU	1264	327	AAAGUUCUUUGCUGCUGCA	1445
  327	UCCCGGCGGGGAGGACCGG	1265	327	UCCCGGCGGGGAGGACCGG	1265	345	CCGGUCCUCCCCGCCGGGA	1446
  345	GAGACAAGUGGCAGAGUCC	1266	345	GAGACAAGUGGCAGAGUCC	1266	363	GGACUCUGCCACUUGUCUC	1447
  363	CCGGAGCGAACUUUUGCAA	1267	363	CCGGAGCGAACUUUUGCAA	1267	381	UUGCAAAAGUUCGCUCCGG	1448
  381	AGCCUUUCCUGCGUCUUAG	1268	381	AGCCUUUCCUGCGUCUUAG	1268	399	CUAAGACGCAGGAAAGGCU	1449
  399	GGCUUCUCCACGGCGGUAA	1269	399	GGCUUCUCCACGGCGGUAA	1269	417	UUACCGCCGUGGAGAAGCC	1450
  417	AAGACCAGAAGGCGGCGGA	1270	417	AAGACCAGAAGGCGGCGGA	1270	435	UCCGCCGCCUUCUGGUCUU	1451
  435	AGAGCCACGCAAGAGAAGA	1271	435	AGAGCCACGCAAGAGAAGA	1271	453	UCUUCUCUUGCGUGGCUCU	1452
  453	AAGGACGUGCGCUCAGCUU	1272	453	AAGGACGUGCGCUCAGCUU	1272	471	AAGCUGAGCGCACGUCCUU	1453
  471	UCGCUCGCACCGGUUGUUG	1273	471	UCGCUCGCACCGGUUGUUG	1273	489	CAACAACCGGUGCGAGCGA	1454
  489	GAACUUGGGCGAGCGCGAG	1274	489	GAACUUGGGCGAGCGCGAG	1274	507	CUCGCGCUCGCCCAAGUUC	1455
  507	GCCGCGGCUGCCGGGCGCC	1275	507	GCCGCGGCUGCCGGGCGCC	1275	525	GGCGCCCGGCAGCCGCGGC	1456
  525	CCCCUCCCCCUAGCAGCGG	1276	525	CCCCUCCCCCUAGCAGCGG	1276	543	CCGCUGCUAGGGGGAGGGG	1457
  543	GAGGAGGGGACAAGUCGUC	1277	543	GAGGAGGGGACAAGUCGUC	1277	561	GACGACUUGUCCCCUCCUC	1458
  561	CGGAGUCCGGGCGGCCAAG	1278	561	CGGAGUCCGGGCGGCCAAG	1278	579	CUUGGCCGCCCGGACUCCG	1459
  579	GACCCGCCGCCGGCCGGCC	1279	579	GACCCGCCGCCGGCCGGCC	1279	597	GGCCGGCCGGCGGCGGGUC	1460
  597	CACUGCAGGGUCCGCACUG	1280	597	CACUGCAGGGUCCGCACUG	1280	615	CAGUGCGGACCCUGCAGUG	1461
  615	GAUCCGCUCCGCGGGGAGA	1281	615	GAUCCGCUCCGCGGGGAGA	1281	633	UCUCCCCGCGGAGCGGAUC	1462
  633	AGCCGCUGCUCUGGGAAGU	1282	633	AGCCGCUGCUCUGGGAAGU	1282	651	ACUUCCCAGAGCAGCGGCU	1463
  651	UGAGUUCGCCUGCGGACUC	1283	651	UGAGUUCGCCUGCGGACUC	1283	669	GAGUCCGCAGGCGAACUCA	1464
  669	CCGAGGAACCGCUGCGCCC	1284	669	CCGAGGAACCGCUGCGCCC	1284	687	GGGCGCAGCGGUUCCUCGG	1465
  687	CGAAGAGCGCUCAGUGAGU	1285	687	CGAAGAGCGCUCAGUGAGU	1285	705	ACUCACUGAGCGCUCUUCG	1466
  705	UGACCGCGACUUUUCAAAG	1286	705	UGACCGCGACUUUUCAAAG	1286	723	CUUUGAAAAGUCGCGGUCA	1467
  723	GCCGGGUAGCGCGCGCGAG	1287	723	GCCGGGUAGCGCGCGCGAG	1287	741	CUCGCGCGCGCUACCCGGC	1468
  741	GUCGACAAGUAAGAGUGCG	1288	741	GUCGACAAGUAAGAGUGCG	1288	759	CGCACUCUUACUUGUCGAC	1469
  759	GGGAGGCAUCUUAAUUAAC	1289	759	GGGAGGCAUCUUAAUUAAC	1289	777	GUUAAUUAAGAUGCCUCCC	1470
  777	CCCUGCGCUCCCUGGAGCG	1290	777	CCCUGCGCUCCCUGGAGCG	1290	795	CGCUCCAGGGAGCGCAGGG	1471
  795	GAGCUGGUGAGGAGGGCGC	1291	795	GAGCUGGUGAGGAGGGCGC	1291	813	GCGCCCUCCUCACCAGCUC	1472
  813	CAGCGGGGACGACAGCCAG	1292	813	CAGCGGGGACGACAGCCAG	1292	831	CUGGCUGUCGUCCCCGCUG	1473
  831	GCGGGUGCGUGCGCUCUUA	1293	831	GCGGGUGCGUGCGCUCUUA	1293	849	UAAGAGCGCACGCACCCGC	1474
  849	AGAGAAACUUUCCCUGUCA	1294	849	AGAGAAACUUUCCCUGUCA	1294	867	UGACAGGGAAAGUUUCUCU	1475
  867	AAAGGCUCCGGGGGGCGCG	1295	867	AAAGGCUCCGGGGGGCGCG	1295	885	CGCGCCCCCCGGAGCCUUU	1476
  885	GGGUGUCCCCCGCUUGCCA	1296	885	GGGUGUCCCCCGCUUGCCA	1296	903	UGGCAAGCGGGGGACACCC	1477
  903	AGAGCCCUGUUGCGGCCCC	1297	903	AGAGCCCUGUUGCGGCCCC	1297	921	GGGGCCGCAACAGGGCUCU	1478
  921	CGAAACUUGUGCGCGCACG	1298	921	CGAAACUUGUGCGCGCACG	1298	939	CGUGCGCGCACAAGUUUCG	1479
  939	GCCAAACUAACCUCACGUG	1299	939	GCCAAACUAACCUCACGUG	1299	957	CACGUGAGGUUAGUUUGGC	1480
  957	GAAGUGACGGACUGUUCUA	1300	957	GAAGUGACGGACUGUUCUA	1300	975	UAGAACAGUCCGUCACUUC	1481
  975	AUGACUGCAAAGAUGGAAA	1301	975	AUGACUGCAAAGAUGGAAA	1301	993	UUUCCAUCUUUGCAGUCAU	1482
  993	ACGACCUUCUAUGACGAUG	1302	993	ACGACCUUCUAUGACGAUG	1302	1011	CAUCGUCAUAGAAGGUCGU	1483
  1011	GCCCUCAACGCCUCGUUCC	1303	1011	GCCCUCAACGCCUCGUUCC	1303	1029	GGAACGAGGCGUUGAGGGC	1484
  1029	CUCCCGUCCGAGAGCGGAC	1304	1029	CUCCCGUCCGAGAGCGGAC	1304	1047	GUCCGCUCUCGGACGGGAG	1485
  1047	CCUUAUGGCUACAGUAACC	1305	1047	CCUUAUGGCUACAGUAACC	1305	1065	GGUUACUGUAGCCAUAAGG	1486
  1065	CCCAAGAUCCUGAAACAGA	1306	1065	CCCAAGAUCCUGAAACAGA	1306	1083	UCUGUUUCAGGAUCUUGGG	1487
  1083	AGCAUGACCCUGAACCUGG	1307	1083	AGCAUGACCCUGAACCUGG	1307	1101	CCAGGUUCAGGGUCAUGCU	1488
  1101	GCCGACCCAGUGGGGAGCC	1308	1101	GCCGACCCAGUGGGGAGCC	1308	1119	GGCUCCCCACUGGGUCGGC	1489
  1119	CUGAAGCCGCACCUCCGCG	1309	1119	CUGAAGCCGCACCUCCGCG	1309	1137	CGCGGAGGUGCGGCUUCAG	1490
  1137	GCCAAGAACUCGGACCUCC	1310	1137	GCCAAGAACUCGGACCUCC	1310	1155	GGAGGUCCGAGUUCUUGGC	1491
  1155	CUCACCUCGCCCGACGUGG	1311	1155	CUCACCUCGCCCGACGUGG	1311	1173	CCACGUCGGGCGAGGUGAG	1492
  1173	GGGCUGCUCAAGCUGGCGU	1312	1173	GGGCUGCUCAAGCUGGCGU	1312	1191	ACGCCAGCUUGAGCAGCCC	1493
  1191	UCGCCCGAGCUGGAGCGCC	1313	1191	UCGCCCGAGCUGGAGCGCC	1313	1209	GGCGCUCCAGCUCGGGCGA	1494
  1209	CUGAUAAUCCAGUCCAGCA	1314	1209	CUGAUAAUCCAGUCCAGCA	1314	1227	UGCUGGACUGGAUUAUCAG	1495
  1227	AACGGGCACAUCACCACCA	1315	1227	AACGGGCACAUCACCACCA	1315	1245	UGGUGGUGAUGUGCCCGUU	1496
  1245	ACGCCGACCCCCACCCAGU	1316	1245	ACGCCGACCCCCACCCAGU	1316	1263	ACUGGGUGGGGGUCGGCGU	1497
  1263	UUCCUGUGCCCCAAGAACG	1317	1263	UUCCUGUGCCCCAAGAACG	1317	1281	CGUUCUUGGGGCACAGGAA	1498
  1281	GUGACAGAUGAGCAGGAGG	1318	1281	GUGACAGAUGAGCAGGAGG	1318	1299	CCUCCUGCUCAUCUGUCAC	1499
  1299	GGGUUCGCCGAGGGCUUCG	1319	1299	GGGUUCGCCGAGGGCUUCG	1319	1317	CGAAGCCCUCGGCGAACCC	1500
  1317	GUGCGCGCCCUGGCCGAAC	1320	1317	GUGCGCGCCCUGGCCGAAC	1320	1335	GUUCGGCCAGGGCGCGCAC	1501
  1335	CUGCACAGCCAGAACACGC	1321	1335	CUGCACAGCCAGAACACGC	1321	1353	GCGUGUUCUGGCUGUGCAG	1502
  1353	CUGCCCAGCGUCACGUCGG	1322	1353	CUGCCCAGCGUCACGUCGG	1322	1371	CCGACGUGACGCUGGGCAG	1503
  1371	GCGGCGCAGCCGGUCAACG	1323	1371	GCGGCGCAGCCGGUCAACG	1323	1389	CGUUGACCGGCUGCGCCGC	1504
  1389	GGGGCAGGCAUGGUGGCUC	1324	1389	GGGGCAGGCAUGGUGGCUC	1324	1407	GAGCCACCAUGCCUGCCCC	1505
  1407	CCCGCGGUAGCCUCGGUGG	1325	1407	CCCGCGGUAGCCUCGGUGG	1325	1425	CCACCGAGGCUACCGCGGG	1506
  1425	GCAGGGGGCAGCGGCAGCG	1326	1425	GCAGGGGGCAGCGGCAGCG	1326	1443	CGCUGCCGCUGCCCCCUGC	1507
  1443	GGCGGCUUCAGCGCCAGCC	1327	1443	GGCGGCUUCAGCGCCAGCC	1327	1461	GGCUGGCGCUGAAGCCGCC	1508
  1461	CUGCACAGCGAGCCGCCGG	1328	1461	CUGCACAGCGAGCCGCCGG	1328	1479	CCGGCGGCUCGCUGUGCAG	1509
  1479	GUCUACGCAAACCUCAGCA	1329	1479	GUCUACGCAAACCUCAGCA	1329	1497	UGCUGAGGUUUGCGUAGAC	1510
  1497	AACUUCAACCCAGGCGCGC	1330	1497	AACUUCAACCCAGGCGCGC	1330	1515	GCGCGCCUGGGUUGAAGUU	1511
  1515	CUGAGCAGCGGCGGCGGGG	1331	1515	CUGAGCAGCGGCGGCGGGG	1331	1533	CCCCGCCGCCGCUGCUCAG	1512
  1533	GCGCCCUCCUACGGCGCGG	1332	1533	GCGCCCUCCUACGGCGCGG	1332	1551	CCGCGCCGUAGGAGGGCGC	1513
  1551	GCCGGCCUGGCCUUUCCCG	1333	1551	GCCGGCCUGGCCUUUCCCG	1333	1569	CGGGAAAGGCCAGGCCGGC	1514
  1569	GCGCAACCCCAGCAGCAGC	1334	1569	GCGCAACCCCAGCAGCAGC	1334	1587	GCUGCUGCUGGGGUUGCGC	1515
  1587	CAGCAGCCGCCGCACCACC	1335	1587	CAGCAGCCGCCGCACCACC	1335	1605	GGUGGUGCGGCGGCUGCUG	1516
  1605	CUGCCCCAGCAGAUGCCCG	1336	1605	CUGCCCCAGCAGAUGCCCG	1336	1623	CGGGCAUCUGCUGGGGCAG	1517
  1623	GUGCAGCACCCGCGGCUGC	1337	1623	GUGCAGCACCCGCGGCUGC	1337	1641	GCAGCCGCGGGUGCUGCAC	1518
  1641	CAGGCCCUGAAGGAGGAGC	1338	1641	CAGGCCCUGAAGGAGGAGC	1338	1659	GCUCCUCCUUCAGGGCCUG	1519
  1659	CCUCAGACAGUGCCCGAGA	1339	1659	CCUCAGACAGUGCCCGAGA	1339	1677	UCUCGGGCACUGUCUGAGG	1520
  1677	AUGCCCGGCGAGACACCGC	1340	1677	AUGCCCGGCGAGACACCGC	1340	1695	GCGGUGUCUCGCCGGGCAU	1521
  1695	CCCCUGUCCCCCAUCGACA	1341	1695	CCCCUGUCCCCCAUCGACA	1341	1713	UGUCGAUGGGGGACAGGGG	1522
  1713	AUGGAGUCCCAGGAGCGGA	1342	1713	AUGGAGUCCCAGGAGCGGA	1342	1731	UCCGCUCCUGGGACUCCAU	1523
  1731	AUCAAGGCGGAGAGGAAGC	1343	1731	AUCAAGGCGGAGAGGAAGC	1343	1749	GCUUCCUCUCCGCCUUGAU	1524
  1749	CGCAUGAGGAACCGCAUCG	1344	1749	CGCAUGAGGAACCGCAUCG	1344	1767	CGAUGCGGUUCCUCAUGCG	1525
  1767	GCUGCCUCCAAGUGCCGAA	1345	1767	GCUGCCUCCAAGUGCCGAA	1345	1785	UUCGGCACUUGGAGGCAGC	1526
  1785	AAAAGGAAGCUGGAGAGAA	1346	1785	AAAAGGAAGCUGGAGAGAA	1346	1803	UUCUCUCCAGCUUCCUUUU	1527
  1803	AUCGCCCGGCUGGAGGAAA	1347	1803	AUCGCCCGGCUGGAGGAAA	1347	1821	UUUCCUCCAGCCGGGCGAU	1528
  1821	AAAGUGAAAACCUUGAAAG	1348	1821	AAAGUGAAAACCUUGAAAG	1348	1839	CUUUCAAGGUUUUCACUUU	1529
  1839	GCUCAGAACUCGGAGCUGG	1349	1839	GCUCAGAACUCGGAGCUGG	1349	1857	CCAGCUCCGAGUUCUGAGC	1530
  1857	GCGUCCACGGCCAACAUGC	1350	1857	GCGUCCACGGCCAACAUGC	1350	1875	GCAUGUUGGCCGUGGACGC	1531
  1875	CUCAGGGAACAGGUGGCAC	1351	1875	CUCAGGGAACAGGUGGCAC	1351	1893	GUGCCACCUGUUCCCUGAG	1532
  1893	CAGCUUAAACAGAAAGUCA	1352	1893	CAGCUUAAACAGAAAGUCA	1352	1911	UGACUUUCUGUUUAAGCUG	1533
  1911	AUGAACCACGUUAACAGUG	1353	1911	AUGAACCACGUUAACAGUG	1353	1929	CACUGUUAACGUGGUUCAU	1534
  1929	GGGUGCCAACUCAUGCUAA	1354	1929	GGGUGCCAACUCAUGCUAA	1354	1947	UUAGCAUGAGUUGGCACCC	1535
  1947	ACGCAGCAGUUGCAAACAU	1355	1947	ACGCAGCAGUUGCAAACAU	1355	1965	AUGUUUGCAACUGCUGCGU	1536
  1965	UUUUGAAGAGAGACCGUCG	1356	1965	UUUUGAAGAGAGACCGUCG	1356	1983	CGACGGUCUCUCUUCAAAA	1537
  1983	GGGGGCUGAGGGGCAACGA	1357	1983	GGGGGCUGAGGGGCAACGA	1357	2001	UCGUUGCCCCUCAGCCCCC	1538
  2001	AAGAAAAAAAAUAACACAG	1358	2001	AAGAAAAAAAAUAACACAG	1358	2019	CUGUGUUAUUUUUUUUCUU	1539
  2019	GAGAGACAGACUUGAGAAC	1359	2019	GAGAGACAGACUUGAGAAC	1359	2037	GUUCUCAAGUCUGUCUCUC	1540
  2037	CUUGACAAGUUGCGACGGA	1360	2037	CUUGACAAGUUGCGACGGA	1360	2055	UCCGUCGCAACUUGUCAAG	1541
  2055	AGAGAAAAAAGAAGUGUCC	1361	2055	AGAGAAAAAAGAAGUGUCC	1361	2073	GGACACUUCUUUUUUCUCU	1542
  2073	CGAGAACUAAAGCCAAGGG	1362	2073	CGAGAACUAAAGCCAAGGG	1362	2091	CCCUUGGCUUUAGUUCUCG	1543
  2091	GUAUCCAAGUUGGACUGGG	1363	2091	GUAUCCAAGUUGGACUGGG	1363	2109	CCCAGUCCAACUUGGAUAC	1544
  2109	GUUCGGUCUGACGGCGCCC	1364	2109	GUUCGGUCUGACGGCGCCC	1364	2127	GGGCGCCGUCAGACCGAAC	1545
  2127	CCCAGUGUGCACGAGUGGG	1365	2127	CCCAGUGUGCACGAGUGGG	1365	2145	CCCACUCGUGCACACUGGG	1546
  2145	GAAGGACUUGGUCGCGCCC	1366	2145	GAAGGACUUGGUCGCGCCC	1366	2163	GGGCGCGACCAAGUCCUUC	1547
  2163	CUCCCUUGGCGUGGAGCCA	1367	2163	CUCCCUUGGCGUGGAGCCA	1367	2181	UGGCUCCACGCCAAGGGAG	1548
  2181	AGGGAGCGGCCGCCUGCGG	1368	2181	AGGGAGCGGCCGCCUGCGG	1368	2199	CCGCAGGCGGCCGCUCCCU	1549
  2199	GGCUGCCCCGCUUUGCGGA	1369	2199	GGCUGCCCCGCUUUGCGGA	1369	2217	UCCGCAAAGCGGGGCAGCC	1550
  2217	ACGGGCUGUCCCCGCGCGA	1370	2217	ACGGGCUGUCCCCGCGCGA	1370	2235	UCGCGCGGGGACAGCCCGU	1551
  2235	AACGGAACGUUGGACUUUC	1371	2235	AACGGAACGUUGGACUUUC	1371	2253	GAAAGUCCAACGUUCCGUU	1552
  2253	CGUUAACAUUGACCAAGAA	1372	2253	CGUUAACAUUGACCAAGAA	1372	2271	UUCUUGGUCAAUGUUAACG	1553
  2271	ACUGCAUGGACCUAACAUU	1373	2271	ACUGCAUGGACCUAACAUU	1373	2289	AAUGUUAGGUCCAUGCAGU	1554
  2289	UCGAUCUCAUUCAGUAUUA	1374	2289	UCGAUCUCAUUCAGUAUUA	1374	2307	UAAUACUGAAUGAGAUCGA	1555
  2307	AAAGGGGGGAGGGGGAGGG	1375	2307	AAAGGGGGGAGGGGGAGGG	1375	2325	CCCUCCCCCUCCCCCCUUU	1556
  2325	GGGUUACAAACUGCAAUAG	1376	2325	GGGUUACAAACUGCAAUAG	1376	2343	CUAUUGCAGUUUGUAACCC	1557
  2343	GAGACUGUAGAUUGCUUCU	1377	2343	GAGACUGUAGAUUGCUUCU	1377	2361	AGAAGCAAUCUACAGUCUC	1558
  2361	UGUAGUACUCCUUAAGAAC	1378	2361	UGUAGUACUCCUUAAGAAC	1378	2379	GUUCUUAAGGAGUACUACA	1559
  2379	CACAAAGCGGGGGGAGGGU	1379	2379	CACAAAGCGGGGGGAGGGU	1379	2397	ACCCUCCCCCCGCUUUGUG	1560
  2397	UUGGGGAGGGGCGGCAGGA	1380	2397	UUGGGGAGGGGCGGCAGGA	1380	2415	UCCUGCCGCCCCUCCCCAA	1561
  2415	AGGGAGGUUUGUGAGAGCG	1381	2415	AGGGAGGUUUGUGAGAGCG	1381	2433	CGCUCUCACAAACCUCCCU	1562
  2433	GAGGCUGAGCCUACAGAUG	1382	2433	GAGGCUGAGCCUACAGAUG	1382	2451	CAUCUGUAGGCUCAGCCUC	1563
  2451	GAACUCUUUCUGGCCUGCU	1383	2451	GAACUCUUUCUGGCCUGCU	1383	2469	AGCAGGCCAGAAAGAGUUC	1564
  2469	UUUCGUUAACUGUGUAUGU	1384	2469	UUUCGUUAACUGUGUAUGU	1384	2487	ACAUACACAGUUAACGAAA	1565
  2487	UACAUAUAUAUAUUUUUUA	1385	2487	UACAUAUAUAUAUUUUUUA	1385	2505	UAAAAAAUAUAUAUAUGUA	1566
  2505	AAUUUGAUUAAAGCUGAUU	1386	2505	AAUUUGAUUAAAGCUGAUU	1386	2523	AAUCAGCUUUAAUCAAAUU	1567
  2523	UACUGUCAAUAAACAGCUU	1387	2523	UACUGUCAAUAAACAGCUU	1387	2541	AAGCUGUUUAUUGACAGUA	1568
  2541	UCAUGCCUUUGUAAGUUAU	1388	2541	UCAUGCCUUUGUAAGUUAU	1388	2559	AUAACUUACAAAGGCAUGA	1569
  2559	UUUCUUGUUUGUUUGUUUG	1389	2559	UUUCUUGUUUGUUUGUUUG	1389	2577	CAAACAAACAAACAAGAAA	1570
  2577	GGGUAUCCUGCCCAGUGUU	1390	2577	GGGUAUCCUGCCCAGUGUU	1390	2595	AACACUGGGCAGGAUACCC	1571
  2595	UGUUUGUAAAUAAGAGAUU	1391	2595	UGUUUGUAAAUAAGAGAUU	1391	2613	AAUCUCUUAUUUACAAACA	1572
  2613	UUGGAGCACUCUGAGUUUA	1392	2613	UUGGAGCACUCUGAGUUUA	1392	2631	UAAACUCAGAGUGCUCCAA	1573
  2631	ACCAUUUGUAAUAAAGUAU	1393	2631	ACCAUUUGUAAUAAAGUAU	1393	2649	AUACUUUAUUACAAAUGGU	1574
  2649	UAUAAUUUUUUUAUGUUUU	1394	2649	UAUAAUUUUUUUAUGUUUU	1394	2667	AAAACAUAAAAAAAUUAUA	1575
  2667	UGUUUCUGAAAAUUCCAGA	1395	2667	UGUUUCUGAAAAUUCCAGA	1395	2685	UCUGGAAUUUUCAGAAACA	1576
  2685	AAAGGAUAUUUAAGAAAAU	1396	2685	AAAGGAUAUUUAAGAAAAU	1396	2703	AUUUUCUUAAAUAUCCUUU	1577
  2703	UACAAUAAACUAUUGGAAA	1397	2703	UACAAUAAACUAUUGGAAA	1397	2721	UUUCCAAUAGUUUAUUGUA	1578
  2721	AGUACUCCCCUAACCUCUU	1398	2721	AGUACUCCCCUAACCUCUU	1398	2739	AAGAGGUUAGGGGAGUACU	1579
  2739	UUUCUGCAUCAUCUGUAGA	1399	2739	UUUCUGCAUCAUCUGUAGA	1399	2757	UCUACAGAUGAUGCAGAAA	1580
  2757	AUCCUAGUCUAUCUAGGUG	1400	2757	AUCCUAGUCUAUCUAGGUG	1400	2775	CACCUAGAUAGACUAGGAU	1581
  2775	GGAGUUGAAAGAGUUAAGA	1401	2775	GGAGUUGAAAGAGUUAAGA	1401	2793	UCUUAACUCUUUCAACUCC	1582
  2793	AAUGCUCGAUAAAAUCACU	1402	2793	AAUGCUCGAUAAAAUCACU	1402	2811	AGUGAUUUUAUCGAGCAUU	1583
  2811	UCUCAGUGCUUCUUACUAU	1403	2811	UCUCAGUGCUUCUUACUAU	1403	2829	AUAGUAAGAAGCACUGAGA	1584
  2829	UUAAGCAGUAAAAACUGUU	1404	2829	UUAAGCAGUAAAAACUGUU	1404	2847	AACAGUUUUUACUGCUUAA	1585
  2847	UCUCUAUUAGACUUAGAAA	1405	2847	UCUCUAUUAGACUUAGAAA	1405	2865	UUUCUAAGUCUAAUAGAGA	1586
  2865	AUAAAUGUACCUGAUGUAC	1406	2865	AUAAAUGUACCUGAUGUAC	1406	2883	GUACAUCAGGUACAUUUAU	1587
  2883	CCUGAUGCUAUGUCAGGCU	1407	2883	CCUGAUGCUAUGUCAGGCU	1407	2901	AGCCUGACAUAGCAUCAGG	1588
  2901	UUCAUACUCCACGCUCCCC	1408	2901	UUCAUACUCCACGCUCCCC	1408	2919	GGGGAGCGUGGAGUAUGAA	1589
  2919	CCAGCGUAUCUAUAUGGAA	1409	2919	CCAGCGUAUCUAUAUGGAA	1409	2937	UUCCAUAUAGAUACGCUGG	1590
  2937	AUUGCUUACCAAAGGCUAG	1410	2937	AUUGCUUACCAAAGGCUAG	1410	2955	CUAGCCUUUGGUAAGCAAU	1591
  2955	GUGCGAUGUUUCAGGAGGC	1411	2955	GUGCGAUGUUUCAGGAGGC	1411	2973	GCCUCCUGAAACAUCGCAC	1592
  2973	CUGGAGGAAGGGGGGUUGC	1412	2973	CUGGAGGAAGGGGGGUUGC	1412	2991	GCAACCCCCCUUCCUCCAG	1593
  2991	CAGUGGAGAGGGACAGCCC	1413	2991	CAGUGGAGAGGGACAGCCC	1413	3009	GGGCUGUCCCUCUCCACUG	1594
  3009	CACUGAGAAGUCAAACAUU	1414	3009	CACUGAGAAGUCAAACAUU	1414	3027	AAUGUUUGACUUCUCAGUG	1595
  3027	UUCAAAGUUUGGAUUGCAU	1415	3027	UUCAAAGUUUGGAUUGCAU	1415	3045	AUGCAAUCCAAACUUUGAA	1596
  3045	UCAAGUGGCAUGUGCUGUG	1416	3045	UCAAGUGGCAUGUGCUGUG	1416	3063	CACAGCACAUGCCACUUGA	1597
  3063	GACCAUUUAUAAUGUUAGA	1417	3063	GACCAUUUAUAAUGUUAGA	1417	3081	UCUAACAUUAUAAAUGGUC	1598
  3081	AAAUUUUACAAUAGGUGCU	1418	3081	AAAUUUUACAAUAGGUGCU	1418	3099	AGCACCUAUUGUAAAAUUU	1599
  3099	UUAUUCUCAAAGCAGGAAU	1419	3099	UUAUUCUCAAAGCAGGAAU	1419	3117	AUUCCUGCUUUGAGAAUAA	1600
  3117	UUGGUGGCAGAUUUUACAA	1420	3117	UUGGUGGCAGAUUUUACAA	1420	3135	UUGUAAAAUCUGCCACCAA	1601
  3135	AAAGAUGUAUCCUUCCAAU	1421	3135	AAAGAUGUAUCCUUCCAAU	1421	3153	AUUGGAAGGAUACAUCUUU	1602
  3153	UUUGGAAUCUUCUCUUUGA	1422	3153	UUUGGAAUCUUCUCUUUGA	1422	3171	UCAAAGAGAAGAUUCCAAA	1603
  3171	ACAAUUCCUAGAUAAAAAG	1423	3171	ACAAUUCCUAGAUAAAAAG	1423	3189	CUUUUUAUCUAGGAAUUGU	1604
  3189	GAUGGCCUUUGUCUUAUGA	1424	3189	GAUGGCCUUUGUCUUAUGA	1424	3207	UCAUAAGACAAAGGCCAUC	1605
  3207	AAUAUUUAUAACAGCAUUC	1425	3207	AAUAUUUAUAACAGCAUUC	1425	3225	GAAUGCUGUUAUAAAUAUU	1606
  3225	CUGUCACAAUAAAUGUAUU	1426	3225	CUGUCACAAUAAAUGUAUU	1426	3243	AAUACAUUUAUUGUGACAG	1607
  3234	UAAAUGUAUUCAAAUACCA	1427	3234	UAAAUGUAUUCAAAUACCA	1427	3252	UGGUAUUUGAAUACAUUUA	1608
  The 3-ends of the Upper sequence and the Lower sequence of the siNA construct can include an overhang sequence, for example about 1, 2, 3, or 4 nucleotides in length, preferably 2 nucleotides in length, wherein the overhanging sequence of the lower sequence is optionally complementary to a portion of the target sequence.
  The upper sequence is also referred to as the sense strand, whereas the lower sequence is also referred to as the antisense strand.
  The upper and lower sequences in the Table can further comprise a chemical modification having Formulae I-VII or any combination thereof.

• TABLE III
  MAP Kinase Synthetic Modified siNA constructs

  Target		Seq			Seq
  Pos	Target	ID	Aliases	Sequence	ID

  MAPK1/ERK2

  3302	ACCAGACCUACUGCCAGAGAACC	1113	MAPK1:424U21 siRNA sense	CAGACCUACUGCCAGAGAATT	1129
  3852	AUCACACAGGGUUCCUGACAGAA	1114	MAPK1:778U21 siRNA sense	CACACAGGGUUCCUGACAGTT	1130
  3892	UUGGCUCUAGUCACUGGCAUCUC	1115	MAPK1:1718U21 siRNA sense	GGCUCUAGUCACUGGCAUCTT	1131
  3946	ACUGUGGAGUUGACUCGGUGUUC	1116	MAPK1:2525U21 siRNA sense	UGUGGAGUUGACUCGGUGUTT	1132
  3302	ACCAGACCUACUGCCAGAGAACC	1113	MAPK1:442L21 siRNA (424C)	UUCUCUGGCAGUAGGUCUGTT	1133
  			antisense
  3852	AUCACACAGGGUUCCUGACAGAA	1114	MAPK1:796L21 siRNA (778C)	CUGUCAGGAACCCUGUGUGTT	1134
  			antisense
  3892	UUGGCUCUAGUCACUGGCAUCUC	1115	MAPK1:1736L21 siRNA (1718C)	GAUGCCAGUGACUAGAGCCTT	1135
  			antisense
  3946	ACUGUGGAGUUGACUCGGUGUUC	1116	MAPK1:2543L21 siRNA (2525C)	ACACCGAGUCAACUCCACATT	1136
  			antisense
  3302	ACCAGACCUACUGCCAGAGAACC	1113	MAPK1:424U21 siRNA stab04	B cAGAccuAcuGccAGAGAATT B	1137
  			sense RPI 30817
  3852	AUCACACAGGGUUCCUGACAGAA	1114	MAPK1:778U21 siRNA stab04	B cAcAcAGGGuuccuGAcAGTT B	1138
  			sense RPI 30818
  3892	UUGGCUCUAGUCACUGGCAUCUC	1115	MAPK1:1718U21 siRNA stab04	B GGcucuAGucAcuGGcAucTT B	1139
  			sense RPI 30819
  3946	ACUGUGGAGUUGACUCGGUGUUC	1116	MAPK1:2525U21 siRNA stab04	B uGuGGAGuuGAcucGGuGuTT B	1140
  			sense RPI 30820
  3302	ACCAGACCUACUGCCAGAGAACC	1113	MAPK1:442L21 siRNA (424C)	uucucuGGcAGuAGGucuGTsT	1141
  			stab05 antisense RPI 30821
  3852	AUCACACAGGGUUCCUGACAGAA	1114	MAPK1:796L21 siRNA (778C)	cuGucAGGAAcccuGuGuGTsT	1142
  			stab05 antisense RPI 30822
  3892	UUGGCUCUAGUCACUGGCAUCUC	1115	MAPK1:1736L21 siRNA (1718C)	GAuGccAGuGAcuAGAGccTsT	1143
  			stab05 antisense RPI 30823
  3946	ACUGUGGAGUUGACUCGGUGUUC	1116	MAPK1:2543L21 siRNA (2525C)	AcAccGAGucAAcuCCAcATsT	1144
  			stab05 antisense RPI 30824
  3302	ACCAGACCUA0UGCCAGAGAACC	1113	MARK1:424U21 siRNA stab07	B cAGAccuAcuGccAGAGAATT B	1145
  			sense
  3852	AUCACACAGGGUUCCUGACAGAA	1114	MARK1:778U21 siRNA stabo7	B cAcAcAGGGuuccuGAcAGTT B	1146
  			sense
  3892	UUGGCUCUAGUCACUGGCAUCUC	1115	MAPK1:1718U21 siRNA stabo7	BGGcucuAGucAcuGGcAucTTB 1147
  			sense
  3946	ACUGUGGAGUUGACUCGGUGUUC	1116	MARK1:2525U21 siRNA stabo7	B uGuGGAGuuGAcucGGuGuTT B	1148
  			sense
  3302	ACCAGACCUACUGCCAGAGAACC	1113	MARK1:442L21 siRNA (424C)	uucucuGGcAGuAGGucuGTsT	1149
  			stab11 antisense
  3852	AUCACACAGGGUUCCUGACAGAA	1114	MARK1:796L21 siRNA (778C)	cuGucAGGAAcccuGuGuGTsT	1150
  			stab11 antisense
  3892	UUGGCUCUAGUCACUGGCAUCUC	1115	MARK1:1736L21 siRNA (1718C)	GAuGccAGuGAcuAGAGccTsT	1151
  			stab11 antisense
  3946	ACUGUGGAGUUGACUCGGUGUUC	1116	MARK1:2543L21 siRNA (25250)	AcAccGAGucAAcuccAcATsT	1152
  			stab11 antisense

  Target		Seq			Seq
  Pos	Target	ID	Aliases	Sequence	ID

  MAPK3/ERK1

  283	CCUUCGAACAUCAGACCUACUGC	1117	MAPK3:285U21 siRNA sense	UUCGAACAUCAGACCUACUTT	1153
  709	UGAACUCCAAGGGCUAUACCAAG	1118	MAPK3:711U21 siRNA sense	AACUCCAAGGGCUAUACCATT	1154
  718	AGGGCUAUACCAAGUCCAUCGAC	1119	MAPK3:720U21 siRNA sense	GGCUAUACCAAGUCCAUCGTT	1155
  1778	UUCUGUGUGUGGUGAGCAGAAGU	1120	MAPK3:1780U21 siRNA sense	CUGUGUGUGGUGAGCAGAATT	1156
  283	CCUUCGAACAUCAGACCUACUGC	1117	MAPK3:303L21 siRNA (285C)	AGUAGGUCUGAUGUUCGAATT	1157
  			antisense
  709	UGAACUCCAAGGGCUAUACCAAG	1118	MAPK3:729L21 siRNA (711C)	UGGUAUAGCCCUUGGAGUUTT	1158
  			antisense
  718	AGGGCUAUACCAAGUCCAUCGAC	1119	MAPK3:738L21 siRNA (720C)	CGAUGGACUUGGUAUAGCCTT	1159
  			antisense
  1778	UUCUGUGUGUGGUGAGCAGAAGU	1120	MAPK3:1798L21 siRNA (1780C)	UUCUGCUCACCACACACAGTT	1160
  			antisense
  283	CCUUCGAACAUCAGACCUACUGC	1117	MAPK3:285U21 siRNA stab04 sense	B uucGAAcAucAGAccuAcuTT B	1161
  709	UGAACUCCAAGGGCUAUACCAAG	1118	MAPK3:711U21 siRNA stab04 sense	B AAcuccAAGGGcuAuAccATT B	1162
  718	AGGGCUAUACCAAGUCCAUCGAC	1119	MAPK3:720U21 siRNA stab04 sense	B GGcuAuAccAAGuccAucGTT B	1163
  1778	UUCUGUGUGUGGUGAGCAGAAGU	1120	MAPK3:1780U21 siRNA stab04 sense	B cuGuGuGuGGuGAGcAGAATT B	1164
  283	CCUUCGAACAUCAGACCUACUGC	1117	MAPK3:303L21 siRNA (285C) stab05	AGuAGGucuGAuGuucGAATsT	1165
  			antisense
  709	UGAACUCCAAGGGCUAUACCAAG	1118	MAPK3:729L21 siRNA (711C) stab05	uGGuAuAGcccuuGGAGuuTsT	1166
  			antisense
  718	AGGGCUAUACCAAGUCCAUCGAC	1119	MAPK3:738L21 siRNA (720C) stab05	cGAuGGAcuuGGuAuAGccTsT	1167
  			antisense
  1778	UUCUGUGUGUGGUGAGCAGAAGU	1120	MAPK3:1798L21 siRNA (1780C)	uucuGcucAccAcAcAcAGTsT	1168
  			stab05 antisense
  283	CCUUCGAACAUCAGACCUACUGC	1117	MAPK3:285U21 siRNA stab07 sense	B uucGAAcAucAGAccuAcuTT B	1169
  709	UGAACUCCAAGGGCUAUACCAAG	1118	MAPK3:711U21 siRNA stab07 sense	BAAcuccAAGGGcuAuAccATT B	1170
  718	AGGGCUAUACCAAGUCCAUCGAC	1119	MAPK3:720U21 siRNA stab07 sense	BGGcuAuAccAAGuccAucGTT B	1171
  1778	UUCUGUGUGUGGUGAGCAGAAGU	1120	MAPK3:1780U21 siRNA stab07 sense	B cuGuGuGuGGuGAGcAGAATT B	1172
  283	CCUUCGAACAUCAGACCUACUGC	1117	MAPK3:303L21 siRNA (285C) stab11	AGuAGGucuGAuGuucGAATsT	1173
  			antisense
  709	UGAACUCCAAGGGCUAUACCAAG	1118	MAPK3:729L21 siRNA (711C) stab11	uGGuAuAGcccuuGGAGuuTsT	1174
  			antisense
  718	AGGGCUAUACCAAGUCCAUCGAC	1119	MAPK3:738L21 siRNA (720C) stab11	cGAuGGAcuuGGuAuAGccTsT	1175
  			antisense
  1778	UUCUGUGUGUGGUGAGCAGAAGU	1120	MAPK3:1798L21 siRNA (178C0)	uucuGcucAccAcAcAcAGTsT	1176
  			stab11 antisense

  Target		Seq				Seq
  Pos	Target	ID	RPI #	Aliases	Sequence	ID

  MAPK8/JNK1

  733	AACAGCUUGGAACACCAUGUCCU	1121	31517	MAPK8:735U21 siRNA sense	CAGCUUGGAACACCAUGUCTT	1177
  853	UUUUCCCAGCUGACUCAGAACAC	1122	31518	MAPK8:855U21 siRNA sense	UUCCCAGCUGACUCAGAACTT	1178
  1224	CAAUGUCAACAGAUCCGACUUUG	1123	31519	MAPK8:1226U21 siRNA sense	AUGUCAACAGAUCCGACUUTT	1179
  1242	CUUUGGCCUCUGAUACAGACAGC	1124	31520	MAPK8:1244U21 siRNA sense	UUGGCCUCUGAUACAGACATT	1180
  733	AACAGCUUGGAACACCAUGUCCU	1121	31521	MAPK8:753L21 siRNA (735C)	GACAUGGUGUUCCAAGCUGTT	1181
  				antisense
  853	UUUUCCCAGCUGACUCAGAACAC	1122	31522	MAPK8:873L21 siRNA (855C)	GUUCUGAGUCAGCUGGGAATT	1182
  				antisense
  1224	CAAUGUCAACAGAUCCGACUUUG	1123	31523	MAPK8:1244L21 siRNA (1226C)	AAGUCGGAUCUGUUGACAUTT	1183
  				antisense
  1242	CUUUGGCCUCUGAUACAGACAGC	1124	31524	MAPK8:1262L21 siRNA (1244C)	UGUCUGUAUCAGAGGCCAATT	1184
  				antisense
  733	AACAGCUUGGAACACCAUGUCCU	1121		MAPK8:735U21 siRNA stab4	B cAGcuuGGAAcAccAuGucTT B	1185
  				sense
  853	UUUUCCCAGCUGACUCAGAACAC	1122		MAPK8:855U21 siRNA stab4	B uucccAGcuGAcucAGAAcTT B	1186
  				sense
  1224	CAAUGUCAACAGAUCCGACUUUG	1123		MAPK8:1226U21 siRNA stab4	BAuGucAAcAGAuccGAcuuTTB	1187
  				sense
  1242	CUUUGGCCUCUGAUACAGACAGC	1124		MAPK8:1244U21 siRNA stab4	B uuGGccucuGAuAcAGAcATT B	1188
  				sense
  733	AACAGCUUGGAACACCAUGUCCU	1121		MAPK8:753L21 siRNA (735C)	GAcAuGGuGuuccAAGcuGTsT	1189
  				stab5 antisense
  853	UUUUCCCAGCUGACUCAGAACAC	1122		MAPK8:873L21 siRNA (855C)	GuucuGAGucAGcuGGGAATsT	1190
  				stab5 antisense
  1224	CAAUGUCAACAGAUCCGACUUUG	1123		MAPK8:1244L21 siRNA (1226C)	AAGucGGAucuGuuGAcAuTsT	1191
  				stab5 antisense
  1242	CUUUGGCCUCUGAUACAGACAGC	1124		MAPK8:1262L21 siRNA (1244C)	uGucuGuAucAGAGGccAATsT	1192
  				stab5 antisense
  733	AACAGCUUGGAACACCAUGUCCU	1121		MAPK8:735U21 siRNA stab7	BcAGcuuGGAAcAccAuGucTTB	1193
  				sense
  853	UUUUCCCAGCUGACUCAGAACAC	1122		MAPK8:855U21 siRNA stab7	B uucccAGcuGAcucAGAAcTT B	1194
  				sense
  1224	CAAUGUCAACAGAUCCGACUUUG	1123		MAPK8:1226U21 siRNA stab7	BAuGucAAcAGAuccGAcuuTT B	1195
  				sense
  1242	CUUUGGCCUCUGAUACAGACAGC	1124		MAPK8:1244U21 siRNA stab7	B uuGGccucuGAuAcAGAcATT B	1196
  				sense
  733	AACAGCUUGGAACACCAUGUCCU	1121		MAPK8:753L21 siRNA (735C)	GAcAuGGuGuuccAAGcuGTsT	1197
  				stab11 antisense
  853	UUUUCCCAGCUGACUCAGAACAC	1122		MAPK8:873L21 siRNA (855C)	GuucuGAGucAGcuGGGAATsT	1198
  				stab11 antisense
  1224	CAAUGUCAACAGAUCCGACUUUG	1123		MAPK8:1244L21 siRNA (1226C)	AAGucGGAucuGuuGAcAuTsT	1199
  				stab11 antisense
  1242	CUUUGGCCUCUGAUACAGACAGC	1124		MAPK8:1262L21 siRNA (1244C)	uGucuGuAucAGAGGccAATsT	1200
  				stab11 antisense

  Target		Seq
  Pos	Target	ID	RPI #	Aliases	Sequence	ID

  MAPK14/p38

  1278	GCCUACUUUGCUCAGUACCACGA	1125	31586	MAPK14:1280U21 siRNA sense	CUACUUUGCUCAGUACCACTT	1201
  1609	UGUCUGUCUUUGUGGGAGGGUAA	1126	31587	MARK14:1611U21 siRNA sense	UCUGUCUUUGUGGGAGGGUTT	1202
  2882	AAAAGGGUCUUCUUGGCAGCUUA	1127	31588	MAPK14:2884U21 siRNA sense	AAGGGUCUUCUUGGCAGCUTT	1203
  3554	GGACUCUAAGCUGGAGCUCUUGG	1128	31589	MAPK14:3556U21 siRNA sense	ACUCUAAGCUGGAGCUCUUTT	1204
  1278	GCCUACUUUGCUCAGUACCACGA	1125	31590	MAPK14:1298L21 siRNA (1280C)	GUGGUACUGAGCAAAGUAGTT	1205
  				antisense
  1609	UGUCUGUCUUUGUGGGAGGGUAA	1126	31591	MAPK14:1629L21 siRNA (1611C)	ACCCUCCCACAAAGACAGATT	1206
  				antisense
  2882	AAAAGGGUCUUCUUGGCAGCUUA	1127	31592	MARK14:2902L21 siRNA (2884C)	AGCUGCCAAGAAGACCCUUTT	1207
  				antisense
  3554	GGACUCUAAGCUGGAGCUCUUGG	1128	31593	MAPK14:3574L21 siRNA (3556C)	AAGAGCUCCAGCUUAGAGUTT	1208
  				antisense
  1278	GCCUACUUUGCUCAGUACCACGA	1125		MAPK14:1280U21 siRNA stab04	B cuAcuuuGcucAGuAccAcTT B	1209
  				sense
  1609	UGUCUGUCUUUGUGGGAGGGUAA	1126		MARK14:1611U21 siRNA stab04	B ucuGucuuuGuGGGAGGGuTT B	1210
  				sense
  2882	AAAAGGGUCUUCUUGGCAGCUUA	1127		MAPK14:2884U21 siRNA stab04	B AAGGGucuucuuGGcAGcuTT B	1211
  				sense
  3554	GGACUCUAAGCUGGAGCUCUUGG	1128		MAPK14:3556U21 siRNA stab04	B AcucuAAGcuGGAGcucuuTT B	1212
  				sense
  1278	GCCUACUUUGCUCAGUACCACGA	1125		MAPK14:1298L21 siRNA (1280C)	GuGGuAcuGAGcAAAGuAGTsT	1213
  				stab05 antisense
  1609	UGUCUGUCUUUGUGGGAGGGUAA	1126		MARK14:1629L21 siRNA (1611C)	AcccucccAcAAAGAcAGATsT	1214
  				stab05 antisense
  2882	AAAAGGGUCUUCUUGGCAGCUUA	1127		MAPK14:2902L21 siRNA (2884C)	AGcuGccAAGAAGAcccuuTsT	1215
  				stab05 antisense
  3554	GGACUCUAAGCUGGAGCUCUUGG	1128		MARK14:3574L21 siRNA (3556C)	AAGAGcuccAGcuuAGAGuTsT	1216
  				stab05 antisense
  1278	GCCUACUUUGCUCAGUACCACGA	1125		MAPK14:1280U21 siRNA stab07	B cuAcuuuGcucAGuAccAcTT B	1217
  				sense
  1609	UGUCUGUCUUUGUGGGAGGGUAA	1126		MARK14:1611U21 siRNA stab07	B ucuGucuuuGuGGGAGGGuTT B	1218
  				sense
  2882	AAAAGGGUCUUCUUGGCAGCUUA	1127		MAPK14:2884U21 siRNA stab07	BAAGGGucuucuuGGcAGcuTT B	1219
  				sense
  3554	GGACUCUAAGCUGGAGCUCUUGG	1128		MAPK14:3556U21 siRNA stab07	BAcucuAAGcuGGAGcucuuTT B	1220
  				sense
  1278	GCCUACUUUGCUCAGUACCACGA	1125		MAPK14:1298L21 siRNA (1280C)	GuGGuAcuGAGcAAAGuAGTsT	1221
  				stab11 antisense
  1609	UGUCUGUCUUUGUGGGAGGGUAA	1126		MARK14:1629L21 siRNA (1611C)	AcccucccAcAAAGAcAGATsT	1222
  				stab11 antisense
  2882	AAAAGGGUCUUCUUGGCAGCUUA	1127		MAPK14:2902L21 siRNA (2884C)	AGcuGccAAGAAGAcccuuTsT	1223
  				stab11 antisense
  3554	GGACUCUAAGCUGGAGCUCUUGG	1128		MAPK14:3574L21 siRNA (3556C)	AAGAGcuccAGcuuAGAGuTsT	1224
  				stab11 antisense

  Target		Seq				Seq
  Pos	Target	ID	RPI#	Aliases	Sequence	ID

  c-JUN

  1817	GGAAAAAGUGAAAACCUUGAAAG	1609		JUN:1819U21 siRNA sense	AAAAAGUGAAAACCUUGAATT	1617
  1935	CAACUCAUGCUAACGCAGCAGUU	1610		JUN:1937U21 siRNA sense	ACUCAUGCUAACGCAGCAGTT	1618
  2259	CAUUGACCAAGAACUGCAUGGAC	1611		JUN:2261U21 siRNA sense	UUGACCAAGAACUGCAUGGTT	1619
  2264	ACCAAGAACUGCAUGGACCUAAC	1612		JUN:2266U21 siRNA sense	CAAGAACUGCAUGGACCUATT	1620
  2269	GAACUGCAUGGACCUAACAUUCG	1613		JUN:2271U21 siRNA sense	ACUGCAUGGACCUAACAUUTT	1621
  2270	AACUGCAUGGACCUAACAUUCGA	1614		JUN:2272U21 siRNA sense	CUGCAUGGACCUAACAUUCTT	1622
  2272	CUGCAUGGACCUAACAUUCGAUC	1615		JUN:2274U21 siRNA sense	GCAUGGACCUAACAUUCGATT	1623
  2274	GCAUGGACCUAACAUUCGAUCUC	1616		JUN:2276U21 siRNA sense	AUGGACCUAACAUUCGAUCTT	1624
  1817	GGAAAAAGUGAAAACCUUGAAAG	1609		JUN:1837L21 siRNA (1819C)	UUCAAGGUUUUCACUUUUUTT	1625
  				antisense
  1935	CAACUCAUGCUAACGCAGCAGUU	1610		JUN:1955L21 siRNA (1937C)	CUGCUGCGUUAGCAUGAGUTT	1626
  				antisense
  2259	CAUUGACCAAGAACUGCAUGGAC	1611		JUN:2279L21 siRNA (2261C)	CCAUGCAGUUCUUGGUCAATT	1627
  				antisense
  2264	ACCAAGAACUGCAUGGACCUAAC	1612		JUN:2284L21 siRNA (2266C)	UAGGUCCAUGCAGUUCUUGTT	1628
  				antisense
  2269	GAACUGCAUGGACCUAACAUUCG	1613		JUN:2289L21 siRNA (2271C)	AAUGUUAGGUCCAUGCAGUTT	1629
  				antisense
  2270	AACUGCAUGGACCUAACAUUCGA	1614		JUN:2290L21 siRNA (2272C)	GAAUGUUAGGUCCAUGCAGTT	1630
  				antisense
  2272	CUGCAUGGACCUAACAUUCGAUC	1615		JUN:2292L21 siRNA (2274C)	UCGAAUGUUAGGUCCAUGCTT	1631
  				antisense
  2274	GCAUGGACCUAACAUUCGAUCUC	1616		JUN:2294L21 siRNA (2276C)	GAUCGAAUGUUAGGUCCAUTT	1632
  				antisense
  1817	GGAAAAAGUGAAAACCUUGAAAG	1609		JUN:1819U21 siRNA stab04	B AAAAAGuGAAAAccuuGAATT B	1633
  				sense
  1935	CAACUGAUGCUAACGCAGCAGUU	1610		JUN:1937U21 siRNA stab04	B AcucAuGcuAAcGcAGcAGTT B	1634
  				sense
  2259	CAUUGACCAAGAACUGCAUGGAC	1611		JUN:2261U21 siRNA stab04	B uuGAccAAGAAcuGcAuGGTT B	1635
  				sense
  2264	ACCAAGAACUGCAUGGACCUAAC	1612		JUN:2266U21 siRNA stab04	B cAAGAAcuGcAuGGAccuATT B	1636
  				sense
  2269	GAACUGCAUGGACCUAACAUUCG	1613		JUN:2271U21 siRNA stab04	B AcuGcAuGGAccuAAcAuuTT B	1637
  				sense
  2270	AACUGCAUGGACCUAACAUUCGA	1614		JUN:2272U21 siRNA stab04	B cuGcAuGGAccuAAcAuucTT B	1638
  				sense
  2272	CUGCAUGGACCUAACAUUCGAUC	1615		JUN:2274U21 siRNA stab04	B GcAuGGAccuAAcAuucGATT B	1639
  				sense
  2274	GCAUGGACCUAACAUUCGAUCUC	1616		JUN:2276U21 siRNA stab04	B AuGGAccuAAcAuucGAucTT B	1640
  				sense
  1817	GGAAAAAGUGAAAACCUUGAAAG	1609		JUN:1837L21 siRNA (1819C)	uucAAGGuuuucAcuuuuuTsT	1641
  				stab05 antisense
  1935	CAACUCAUGCUAACGCAGCAGUU	1610		JUN:1955L21 siRNA (1937C)	cuGcuGcGuuAGcAuGAGuTsT	1642
  				stab05 antisense
  2259	CAUUGACCAAGAACUGCAUGGAC	1611		JUN:2279L21 siRNA (2261C)	ccAuGcAGuucuuGGucAATsT	1643
  				stab05 antisense
  2264	ACCAAGAACUGCAUGGACCUAAC	1612		JUN:2284L21 siRNA (2266C)	uAGGuccAuGcAGuucuuGTsT	1644
  				stab05 antisense
  2269	GAACUGCAUGGACCUAACAUUCG	1613		JUN:2289L21 siRNA (2271C)	AAuGuuAGGuccAuGcAGuTsT	1645
  				stab05 antisense
  2270	AACUGCAUGGACCUAACAUUCGA	1614		JUN:2290L21 siRNA (2272C)	GAAuGuuAGGuccAuGcAGTsT	1646
  				stab05 antisense
  2272	CUGCAUGGACCUAACAUUCGAUC	1615		JUN:2292L21 siRNA (2274C)	ucGAAuGuuAGGuccAuGcTsT	1647
  				stab05 antisense
  2274	GCAUGGACCUAACAUUCGAUCUG	1616		JUN:2294L21 siRNA (2276C)	GAucGAAuGuuAGGuccAuTsT	1648
  				stab05 antisense
  1817	GGAAAAAGUGAAAACCUUGAAAG	1609	31818	JUN:1819U21 siRNA stab07	BAAAAAGuGAAAAccuuGAATT B	1649
  				sense
  1935	CAACUCAUGCUAACGCAGCAGUU	1610	31819	JUN:1937U21 siRNA stab07	BAcucAuGcuAAcGcAGcAGTT B	1650
  				sense
  2259	CAUUGACCAAGAACUGCAUGGAC	1611	31820	JUN:2261U21 siRNA stab07	B uuGAccAAGAAcuGcAuGGTT B	1651
  				sense
  2264	ACCAAGAACUGCAUGGACCUAAC	1612	31821	JUN:2266U21 siRNA stab07	B cAAGAAcuGcAuGGAccuATT B	1652
  				sense
  2269	GAACUGCAUGGACCUAACAUUCG	1613	31822	JUN:2271U21 siRNA stab07	BAcuGcAuGGAccuAAcAuuTT B	1653
  				sense
  2270	AACUGCAUGGACCUAACAUUCGA	1614	31823	JUN:2272U21 siRNA stab07	B cuGcAuGGAccuAAcAuucTT B	1654
  				sense
  2272	CUGCAUGGACCUAACAUUCGAUC	1615	31824	JUN:2274U21 siRNA stab07	BGcAuGGAccuAAcAuucGATT B	1655
  				sense
  2274	GCAUGGACCUAACAUUCGAUCUG	1616	31825	JUN:2276U21 siRNA stab07	BAuGGAccuAAcAuucGAucTT B	1656
  				sense
  1817	GGAAAAAGUGAAAACCUUGAAAG	1609		JUN:1837L21 siRNA (1819C)	uucAAGGuuuucAcuuuuuTsT	1657
  				stab11 antisense
  1935	CAACUCAUGCUAACGCAGCAGUU	1610		JUN:1955L21 siRNA (1937C)	cuGcuGcGuuAGcAuGAGuTsT	1658
  				stab11 antisense
  2259	CAUUGACCAAGAACUGCAUGGAC	1611		JUN:2279L21 siRNA (2261C)	ccAucAGuucuuGGucAATsT	1659
  				stab11 antisense
  2264	ACCAAGAACUGCAUGGACCUAAC	1612		JUN:2284L21 siRNA (2266C)	uAGGuccAuGcAGuucuuGTsT	1660
  				stab11 antisense
  2269	GAACUGCAUGGACCUAACAUUCG	1613		JUN:2289L21 siRNA (2271C)	AAuGuuAGGuccAuGcAGuTsT	1661
  				stab11 antisense
  2270	AACUGCAUGGACCUAACAUUCGA	1614		JUN:2290L21 siRNA (2272C)	GAAuGuuAGGuccAuGcAGTsT	1662
  				stab11 antisense
  2272	CUGCAUGGACCUAACAUUCGAUC	1615		JUN:2292L21 siRNA (2274C)	ucGAAuGuuAGGuccAuGcTsT	1663
  				stab11 antisense
  2274	GCAUGGACCUAACAUUCGAUCUC	1616		JUN:2294L21 siRNA (2276C)	GAucGAAuGuuAGGuccAuTsT	1664
  				stab11 antisense
  1817	GGAAAAAGUGAAAACCUUGAAAG	1609		JUN:1819U21 siRNA stab08	AAAAAGuGAAAAccuuGAATsT	1665
  				sense
  1935	CAACUCAUGCUAACGCAGCAGUU	1610		JUN:1937U21 siRNA stab08	AcucAuGcuAAcGcAGcAGTsT	1666
  				sense
  2259	CAUUGACCAAGAACUGCAUGGAC	1611		JUN:2261U21 siRNA stab08	uuGAccAAGAAcuGcAuGGTsT	1667
  				sense
  2264	ACCAAGAACUGCAUGGACCUAAC	1612		JUN:2266U21 siRNA stab08	cAAGAAcuGcAuGGAccuATsT	1668
  				sense
  2269	GAACUGCAUGGACCUAACAUUCG	1613		JUN:2271U21 siRNA stab08	AcuGcAuGGAccuAAcAuuTsT	1669
  				sense
  2270	AACUGCAUGGACCUAACAUUCGA	1614		JUN:2272U21 siRNA stab08	cuGcAuGGAccuAAcAuucTsT	1670
  				sense
  2272	CUGCAUGGACCUAACAUUCGAUC	1615		JUN:2274U21 siRNA stabo8	GcAuGGAccuAAcAuucGATsT	1671
  				sense
  2274	GCAUGGACCUAACAUUCGAUCUC	1616		JUN:2276U21 siRNA stabo8	AuGGAccuAAcAuucGAucTsT	1672
  				sense
  1817	GGAAAAAGUGAAAACCUUGAAAG	1609	31826	JUN:1837L21 siRNA (1819C)	uucAAGGuuuucAcuuuuuTsT	1673
  				stab08 antisense
  1935	CAACUCAUGCUAACGCAGCAGUU	1610	31827	JUN:1955L21 siRNA (1937C)	cuGcuGcGuuAGcAuGAGuTsT	1674
  				stab08 antisense
  2259	CAUUGACCAAGAACUGCAUGGAC	1611	31828	JUN:2279L21 siRNA (2261C)	ccAuGcAGuucuuGGucAATsT	1675
  				stab08 antisense
  2264	ACCAAGAACUGCAUGGACCUAAC	1612	31829	JUN:2284L21 siRNA (2266C)	uAGGuccAuGcAGuucuuGTsT	1676
  				stab08 antisense
  2269	GAACUGCAUGGACCUAACAUUCG	1613	31830	JUN:2289L21 siRNA (2271C)	AAuGuuAGGuccAuGcAGuTsT	1677
  				stab08 antisense
  2270	AACUGCAUGGACCUAACAUUCGA	1614	31831	JUN:2290L21 siRNA (2272C)	GAAuGuuAGGuccAuGcAGTsT	1678
  				stab08 antisense
  2272	CUGCAUGGACCUAACAUUCGAUC	1615	31832	JUN:2292L21 siRNA (2274C)	ucGAAuGuuAGGuccAuGcTsT	1679
  				stab08 antisense
  2274	GCAUGGACCUAACAUUCGAUCUC	1616	31833	JUN:2294L21 siRNA (2276C)	GAucGAAuGuuAGGuccAuTsT	1680
  				stab08 antisense
  1817	GGAAAAAGUGAAAACCUUGAAAG	1609	31834	JUN:1819U21 siRNA inv stab07	BAAGuuccAAAAGuGAAAAATT B	1681
  				sense
  1935	CAACUCAUGCUAACGCAGCAGUU	1610	31835	JUN:1937U21 siRNA inv stab07	BGAcGAcGcAAucGuAcucATT B	1682
  				sense
  2259	CAUUGACCAAGAACUGCAUGGAC	1611	31836	JUN:2261U21 siRNA inv stab07	BGGuAcGucAAGAAccAGuuTT B	1683
  				sense
  2264	ACCAAGAACUGCAUGGACCUAAC	1612	31837	JUN:2266U21 siRNA inv stab07	BAuccAGGuAcGucAAGAAcTT B	1684
  				sense
  2269	GAACUGCAUGGACCUAACAUUCG	1613	31838	JUN:2271U21 siRNA inv stab07	B uuAcAAuccAGGuAcGucATT B	1685
  				sense
  2270	AACUGCAUGGACCUAACAUUCGA	1614	31839	JUN:2272U21 siRNA inv stab07	B cuuAcAAuccAGGuAcGucTT B	1686
  				sense
  2272	CUGCAUGGACCUAACAUUCGAUC	1615	31840	JUN:2274U21 siRNA inv stab07	BAGcuuAcAAuccAGGuAcGTT B	1687
  				sense
  2274	GCAUGGACCUAACAUUCGAUCUC	1616	31841	JUN:2276U21 siRNA inv stab07	B cuAGcuuAcAAuccAGGuATT B	1688
  				sense
  1817	GGAAAAAGUGAAAACCUUGAAAG	1609	31842	JUN:1837L21 siRNA (1819C)	uuuuucAcuuuuGGAAcuuTsT	1689
  				inv stab08 antisense
  1935	CAACUCAUGCUAACGCAGCAGUU	1610	31843	JUN:1955L21 siRNA (1937C)	uGAGuAcGAuuGcGucGucTsT	1690
  				inv stab08 antisense
  2259	CAUUGACCAAGAACUGCAUGGAC	1611	31844	JUN:2279L21 siRNA (2261C)	AAcuGGuucuuGAcGuAccTsT	1691
  				inv stab08 antisense
  2264	ACCAAGAACUGCAUGGACCUAAC	1612	31845	JUN:2284L21 siRNA (2266C)	GuucuuGAcGuAccuGGAuTsT	1692
  				inv stab08 antisense
  2269	GAACUGCAUGGACCUAACAUUCG	1613	31846	JUN:2289L21 siRNA (2271C)	uGAcGuAccuGGAuuGuAATsT	1693
  				inv stab08 antisense
  2270	AACUGCAUGGACCUAACAUUCGA	1614	31847	JUN:2290L21 siRNA (2272C)	GAcGuAccuGGAuuGuAAGTsT	1694
  				inv stab08 antisense
  2272	CUGCAUGGACCUAACAUUCGAUC	1615	31848	JUN:2292L21 siRNA (2274C)	cGuAccuGGAuuGuAAGcuTsT	1695
  				inv stab08 antisense
  2274	GCAUGGACCUAACAUUCGAUCUC	1616	31849	JUN:2294L21 siRNA (2276C)	uAccuGGAuuGuAAGcuAGTsT	1696
  				inv stab08 antisense
  Uppercase = ribonucleotide
  u,c = 2′-deoxy-2′-fluoro U, C
  A = 2′-O-methyl Adenosine
  G = 2′-O-methyl Guanosine
  T = thymidine
  B = inverted deoxy abasic
  s = phosphorothioate linkage
  A = deoxy Adenosine
  G = deoxy Guanosine

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

  Chemistry	pyrimidine	Purine	cap	p = S	Strand
  “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′-ends	—	Usually S
  “Stab 5”	2′-fluoro	Ribo	—	1 at 3′-end	Usually AS
  “Stab 6”	2′-O-Methyl	Ribo		5′ and 3′-ends	—	Usually S
  “Stab 7”	2′-fluoro	2′-deoxy	5′ and 3′-ends	—	Usually S
  “Stab 8”	2′-fluoro	2′-O-Methyl	—	1 at 3′-end	S or AS
  “Stab 9”	Ribo	Ribo	5′ and 3′-ends	—	Usually S
  “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′-ends		Usually S
  “Stab 13”	2′-fluoro	LNA			1 at 3′-end	Usually AS
  “Stab 14”	2′-fluoro	2′-deoxy		2 at 5′-end	UsuallyAS
  				1 at 3′-end
  “Stab 15”	2′-deoxy	2′-deoxy		2 at 5′-end	UsuallyAS
  				1 at 3′-end
  “Stab 16	Ribo	2′-O-Methyl	5′ and 3′-ends		Usually S
  “Stab 17”	2′-O-Methyl	2′-O-Methyl	5′ and 3′-ends		Usually S
  “Stab 18”	2′-fluoro	2′-O-Methyl		1 at 3′-end	Usually AS
  CAP = any terminal cap, see for example FIG. 10.
  All Stab 1-18 chemistries can comprise 3′-terminal thymidine (TT) residues
  All Stab 1-18 chemistries typically comprise 21 nucleotides, but can vary as described herein.
  S = sense strand
  AS = antisense strand

• TABLE V
  A. 2.5 μmol Synthesis Cycle ABI 394 Instrument

  Reagent	Equivalents	Amount	Wait Time* DNA	Wait Time* 2′-O-methyl	Wait Time*RNA

  Phosphoramidites	6.5	163	μL	45	sec	2.5	min	7.5	min
  S-Ethyl Tetrazole	23.8	238	μL	45	sec	2.5	min	7.5	min
  Acetic Anhydride	100	233	μL	5	sec	5	sec	5	sec
  N-Methyl	186	233	μL	5	sec	5	sec	5	sec
  Imidazole
  TCA	176	2.3	mL	21	sec	21	sec	21	sec
  Iodine	11.2	1.7	mL	45	sec	45	sec	45	sec
  Beaucage	12.9	645	μL	100	sec	300	sec	300	sec

  Acetonitrile	NA	6.67	mL	NA	NA	NA

  B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

  Reagent	Equivalents	Amount	Wait Time* DNA	Wait Time* 2′-O-methyl	Wait Time*RNA

  Phosphoramidites	15	31	μL	45	sec	233	sec	465	sec
  S-Ethyl Tetrazole	38.7	31	μL	45	sec	233	min	465	sec
  Acetic Anhydride	655	124	μL	5	sec	5	sec	5	sec
  N-Methyl	1245	124	μL	5	sec	5	sec	5	sec
  Imidazole
  TCA	700	732	μL	10	sec	10	sec	10	sec
  Iodine	20.6	244	μL	15	sec	15	sec	15	sec
  Beaucage	7.7	232	μL	100	sec	300	sec	300	sec

  Acetonitrile	NA	2.64	mL	NA	NA	NA

  C. 0.2 μmol Synthesis Cycle 96 well Instrument

  	Equivalents: DNA/	Amount: DNA/2′-O-		Wait Time* 2′-O-
  Reagent	2′-O-methyl/Ribo	methyl/Ribo	Wait Time* DNA	methyl	Wait Time* Ribo

  Phosphoramidites	22/33/66	40/60/120	μL	60	sec	180	sec	360	sec
  S-Ethyl Tetrazole	70/105/210	40/60/120	μL	60	sec	180	min	360	sec
  Acetic Anhydride	265/265/265	50/50/50	μL	10	sec	10	sec	10	sec
  N-Methyl	502/502/502	50/50/50	μL	10	sec	10	sec	10	sec
  Imidazole
  TCA	238/475/475	250/500/500	μL	15	sec	15	sec	15	sec
  Iodine	6.8/6.8/6.8	80/80/80	μL	30	sec	30	sec	30	sec
  Beaucage	34/51/51	80/120/120		100	sec	200	sec	200	sec

  Acetonitrile	NA	1150/1150/1150	μL	NA	NA	NA
  Wait time does not include contact time during delivery.
  Tandem synthesis utilizes double coupling of linker molecule

Claims (20)

1. A chemically modified nucleic acid molecule, wherein:

(a) the nucleic acid molecule comprises a sense strand and a separate antisense strand, each strand having one or more pyrimidine nucleotides and one or more purine nucleotides;

(b) each strand of the nucleic acid molecule is independently 18 to 27 nucleotides in length;

(c) an 18 to 27 nucleotide sequence of the antisense strand is complementary to a human c-Jun RNA sequence comprising SEQ ID NO:1715;

(d) an 18 to 27 nucleotide sequence of the sense strand is complementary to the antisense strand and comprises an 18 to 27 nucleotide sequence of the human RNA sequence; and

(e) 50 percent or more of the nucleotides in at least one strand comprise a 2′-sugar modification, wherein the 2′-sugar modification of any of the pyrimidine nucleotides differs from the 2′-sugar modification of any of the purine nucleotides.

2. The nucleic acid molecule ofclaim 1, wherein 50 percent or more of the nucleotides in each strand comprise a 2′-sugar modification.

3. The nucleic acid molecule ofclaim 1, wherein the 2′-sugar modification is selected from the group consisting of 2′-deoxy-2′-fluoro, 2′-O-methyl, and 2′-deoxy.

4. The nucleic acid ofclaim 3, wherein the 2′-deoxy-2′-fluoro sugar modification is a pyrimidine modification.

5. The nucleic acid ofclaim 3, wherein the 2′-deoxy sugar modification is a pyrimidine modification.

6. The nucleic acid ofclaim 3, wherein the 2′-O-methyl sugar modification is a pyrimidine modification.

7. The nucleic acid molecule ofclaim 4, wherein said pyrimidine modification is in the sense strand, the antisense strand, or both the sense strand and antisense strand.

8. The nucleic acid molecule ofclaim 6, wherein said pyrimidine modification is in the sense strand, the antisense strand, or both the sense strand and antisense strand.

9. The nucleic acid molecule ofclaim 3, wherein the 2′-deoxy sugar modification is a purine modification.

10. The nucleic acid molecule ofclaim 3, wherein the 2′-O-methyl sugar modification is a purine modification.

11. The nucleic acid molecule ofclaim 9, wherein the purine modification is in the sense strand.

12. The nucleic acid molecule ofclaim 10, wherein the purine modification is in the antisense strand.

13. The nucleic acid molecule ofclaim 1, wherein the nucleic acid molecule comprises ribonucleotides.

14. The nucleic acid molecule ofclaim 1, wherein the sense strand includes a terminal cap moiety at the 5′-end, the 3′-end, or both of the 5′- and 3′-ends.

15. The nucleic acid molecule ofclaim 14, wherein the terminal cap moiety is an inverted deoxy abasic moiety.

16. The nucleic acid molecule ofclaim 1, wherein said nucleic acid molecule includes one or more phosphorothioate internucleotide linkages.

17. The nucleic acid molecule ofclaim 16, wherein one of the phosphorothioate internucleotide linkages is at the 3′-end of the antisense strand.

18. The nucleic acid molecule ofclaim 1, wherein the 5′-end of the antisense strand includes a terminal phosphate group.

19. The nucleic acid molecule ofclaim 1, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand include a 3′-overhang.

20. A composition comprising the nucleic acid molecule ofclaim 1, in a pharmaceutically acceptable carrier or diluent.

Images