*US20090023676A1*
  US20090023676A1                                 
(19)United States 
(12)Patent Application Publication(10)Pub. No.: US 2009/0023676 A1
 McSwiggen et al.(43)Pub. Date:Jan.  22, 2009

(54)RNA Interference Mediated Inhibition of MAP Kinase Gene Expression or Expression of Genes Involved in MAP Kinase Pathway Using Short Interfering Nucleic Acid (SiNA) 
    
(75)Inventors: James McSwiggen,  Bothell, WA (US); 
  Leonid Beigelman,  Brisbane, CA (US); 
  Nassim Usman,  Lafayette, CO (US); 
  Peter Haeberli,  Berthoud, CO (US); 
  Bharat M. Chowrira,  Louisville, CO (US); 
  Barry Polisky,  Boulder, CO (US) 
    
 Correspondence Address: 
 MCDONNELL, BOEHNEN, HULBERT AND BERGHOFF, LLP  
 300 SOUTH WACKER DRIVE, SUITE 3100 
 CHICAGO, IL 60606  (US) 
    
(73)Assignee:Sirna Therapeutics, Inc.,  San Francisco, CA (US), Type: US Company 
(21)Appl. No.: 12/201,759 
(22)Filed: Aug.  29, 2008 
 Related U.S. Application Data 
(63) .
Continuation of application No. 10/424,339, filed on Apr.  25, 2003 , which is a continuation-in-part of application No. PCT/US03/02510, filed on Jan.  28, 2003 , which is a continuation-in-part of application No. PCT/US03/05346, filed on Feb.  20, 2003 , which is a continuation-in-part of application No. PCT/US03/05028, filed on Feb.  20, 2003 .
 
(60)Provisional application No. 60/358,580, filed on Feb.  20, 2002.
 
 Provisional application No. 60/363,124, filed on Mar.  11, 2002.
 
 Provisional application No. 60/386,782, filed on Jun.  6, 2002.
 
 Provisional application No. 60/406,784, filed on Aug.  29, 2002.
 
 Provisional application No. 60/408,378, filed on Sep.  5, 2002.
 
 Provisional application No. 60/409,293, filed on Sep.  9, 2002.
 
 Provisional application No. 60/440,129, filed on Jan.  15, 2003.
 
 Publication Classification 
(51)Int. Cl. A61K 031/7088 (20060101); C07H 021/02 (20060101)
(52)U.S. Cl. 514/44; 536/24.5

        

(57)

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).
 Claim(s),  Drawing Sheet(s), and Figure(s)
 
 


[0001] 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

[0002] 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

[0003] 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

[0004] 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.
[0005] 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.
[0006] 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).
[0007] RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. 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 in Drosophila cells 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 in Drosophila embryonic 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).
[0008] 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.
[0009] Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using 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.
[0010] 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 a Drosophila in 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.
[0011] 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 of C. 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 a Neurospora silencing 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 certain Drosophila-derived gene products that may be related to RNAi in Drosophila. 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 certain C. elegans genes 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

[0012] 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 example FIG. 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.
[0013] 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. NM002750), p38 (also referred to as MAPK14, for example Genbank Accession No. NM139012), ERK2 (also referred to as MAPK1, for example Genbank Accession No. NM002745), and ERK1 (also referred to as MAPK3, for example Genbank Accession XM055766) 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. NM002228), 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. NM013277). 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.
[0014] 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).
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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).
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] In one embodiment, the invention features a medicament comprising a siNA molecule of the invention.
[0047] In one embodiment, the invention features an active ingredient comprising a siNA molecule of the invention.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] In one embodiment, the invention features a composition comprising a siNA molecule of the invention and a pharmaceutically acceptable carrier or diluent.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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:
[see pdf for image]
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.
[0057] 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.
[0058] 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:
[see pdf for image]
[0059] 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.
[0060] 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.
[0061] 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:
[see pdf for image]
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.
[0062] 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.
[0063] 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.
[0064] 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:
[see pdf for image]
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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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:
[see pdf for image]
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.
[0077] 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:
[see pdf for image]
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.
[0078] 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:
[see pdf for image]
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.
[0079] 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” (for example modification 6 in FIG. 10).
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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).
[0085] 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.
[0086] 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).
[0087] 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.
[0088] 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).
[0089] 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.
[0090] 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).
[0091] 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).
[0092] 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 in FIG. 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 in FIGS. 4 and 5 and Tables III and IV herein.
[0093] 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 in FIG. 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 in FIGS. 4 and 5 and Tables III and IV herein.
[0094] 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 in FIG. 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 in FIGS. 4 and 5 and Tables III and IV herein.
[0095] 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 in FIG. 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.
[0096] 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.
[0097] 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.
[0098] 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.)
[0099] 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 and Nucleic 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 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 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.
[0100] 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.
[0101] 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.
[0102] 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 in FIG. 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.
[0103] 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 in FIG. 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.
[0104] 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 in FIG. 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.
[0105] 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 in FIG. 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.
[0106] 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 in FIG. 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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).
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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 a terminal 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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).
[0167] 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.
[0168] 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 in FIGS. 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).
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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).
[0173] 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).
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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. USA 83:9373-9377; 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.
[0179] 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.
[0180] 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/or FIGS. 4-5.
[0181] 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.
[0182] 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/or FIGS. 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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).
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
[0197] 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

[0198] FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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 in FIG. 4 A-F, the modified internucleotide linkage is optional.
[0208] FIG. 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in FIG. 4A-F to a c-JUN siNA sequence. Such chemical modifications can be applied to any sequence herein, such as any MAP kinase sequence.
[0209] FIG. 6 shows non-limiting examples of different siNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.
[0210] FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] FIG. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.
[0215] 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).
[0216] FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.
[0222] FIG. 9E: The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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 INVENTION

Mechanism of Action of Nucleic Acid Molecules of the Invention

[0227] 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.
[0228] 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.
[0229] 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.
[0230] RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. 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 in Drosophila cells 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 in Drosophila embryonic 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

[0231] 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.
[0232] 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 Enzymology 211, 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] For purification of the trityl-on oligomers, the quenched NH4.HCO3 solution 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.
[0238] The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic 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.
[0239] 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, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.
[0240] 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.
[0241] 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.
[0242] 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, TIBS 17, 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.
[0243] 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.

[0244] 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., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 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.
[0245] 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.
[0246] 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.
[0247] 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 Enzymology 211, 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.
[0248] 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).
[0249] 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.
[0250] 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.
[0251] The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
[0252] 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.
[0253] 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.
[0254] 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.
[0255] 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.
[0256] 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.
[0257] 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.
[0258] 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.
[0259] 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/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
[0260] 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.
[0261] 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, NO2 or 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, NO2 or N(CH3)2, amino or SH.
[0262] 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.
[0263] 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.
[0264] 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, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.
[0265] 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.
[0266] 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.
[0267] 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.
[0268] In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH2 or 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.
[0269] 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

[0270] 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.
[0271] 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.
[0272] 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.
[0273] 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.
[0274] 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.
[0275] 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.
[0276] 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., Science 1995, 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.
[0277] 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, in Remington'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.
[0278] 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.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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.
[0283] 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
[0284] 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.
[0285] 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.
[0286] 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.
[0287] 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.
[0288] 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.
[0289] 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.
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] 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., USA 83, 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., 1990 Science, 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.
[0295] 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).
[0296] 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).
[0297] 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).
[0298] 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).
[0299] 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.
[0300] 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.
[0301] 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

[0302] 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 example FIG. 12).
[0303] 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.
[0304] 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.
[0305] 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 Thr183 and Tyr185 of 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] Obesity is closely associated with insulin resistance and establishes the leading risk factor for type 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 of type 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.
[0310] 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.
[0311] 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.
[0312] 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

[0313] The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1

Tandem Synthesis of siNA Constructs

[0314] 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.
[0315] 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 a terminal 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.
[0316] 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 (see FIG. 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.
[0317] 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 1 CV 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 1 CV 20% aqueous CAN.
[0318] 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 2

Identification of Potential siNA Target Sites in Any RNA Sequence

[0319] 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 3

Selection of siNA Molecule Target Sites in a RNA

[0320] The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.
[0321] 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.
[0322] 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.
[0323] 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 in case 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.
[0324] 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.
[0325] 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.
[0326] 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.
[0327] 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.
[0328] 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.
[0329] 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.
[0330] 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 in FIG. 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 example FIG. 7 and FIG. 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 4

MAP Kinase Targeted siNA Design

[0331] 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.
[0332] 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 example FIG. 11).

Example 5

Chemical Synthesis and Purification of siNA

[0333] 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).
[0334] 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).
[0335] 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.
[0336] 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 6

RNAi In Vitro Assay to Assess siNA Activity

[0337] 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. A Drosophila extract 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. The Drosophila lysate 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.
[0338] 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.
[0339] 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 7

Nucleic Acid Inhibition of MAP Kinase Target RNA In Vivo

[0340] 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.
[0341] 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
[0342] Cells (e.g., 293, HeLa, or HepG2 cells) are seeded, for example, at 1×105 cells 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 1×103 in 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
[0343] 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

[0344] 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 8

Models Useful to Evaluate the Down-Regulation of MAP Kinase Gene (e.g., c-JUN, ERK1, ERK2, JNK1, JNK2, and/or p38) expression

Cell Culture

[0345] 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.
[0346] 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

[0347] Evaluating the efficacy of anti-MAP kinase agents in animal models is an important prerequisite to human clinical trials. Obesity and type 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).
[0348] 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.
[0349] 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 9

RNAi Mediated Inhibition of p38 RNA Expression

[0350] 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.
[0351] In a non-limiting example, siNA constructs were screened for activity (see FIG. 12) and compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown in FIG. 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 10

RNAi Mediated Inhibition of p38 RNA Expression

[0352] 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.
[0353] In a non-limiting example, siNA constructs were screened for activity (see FIG. 13) and compared to untreated cells, scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown in FIG. 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 11

Indications

[0354] 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.
[0355] 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 example Cancer: 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 12

Diagnostic Uses

[0356] 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).
[0357] 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.
[0358] 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.
[0359] 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.
[0360] 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.
[0361] 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.
[0362] 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.
[00001] [TABLE-US-00001]
  TABLE I
 
  MAP kinase Accession Numbers
 
 
  NM_002745Homo sapiens mitogen-activated protein kinase 1 (MAPK1), transcript variant 1, mRNA.
  NM_138957Homo sapiens mitogen-activated protein kinase 1 (MAPK1), transcript variant 2, mRNA.
  X60188   Human ERK1 mRNA for protein serine/threonine kinase (MAPK3).
  XM_055766Homo sapiens mitogen-activated protein kinase 3 (MAPK3), mRNA
  NM_002747Homo sapiens mitogen-activated protein kinase 4 (MAPK4), mRNA
  XM_165662Homo sapiens Mitogen-activated protein kinase 4 (Extracellular signal-regulated kinase 4) (ERK-4) (MAP kinase
    isoform p63) (p63-MAPK) (LOC220131), mRNA
  NM_002748Homo sapiens mitogen-activated protein kinase 6 (MAPK6), mRNA.
  XM_166057Homo sapiens Mitogen-activated protein kinase 6 (Extracellular signal-regulated kinase 3) (ERK-3) (MAP kinase
    isoform p97) (p97-MAPK) (LOC220839), mRNA
  XM_035575Homo sapiens mitogen-activated protein kinase 6 (MAPK6), mRNA
  NM_139033Homo sapiens mitogen-activated protein kinase 7 (MAPK7), transcript variant 1, mRNA
  NM_139032Homo sapiens mitogen-activated protein kinase 7 (MAPK7), transcript variant 2, mRNA
  NM_002749Homo sapiens mitogen-activated protein kinase 7 (MAPK7), transcript variant 3, mRNA
  NM_139034Homo sapiens mitogen-activated protein kinase 7 (MAPK7), transcript variant 4, mRNA
  NM_139049Homo sapiens mitogen-activated protein kinase 8 (MAPK8), transcript variant 1, mRNA.
  NM_002750Homo sapiens mitogen-activated protein kinase 8 (MAPK8), transcript variant 2, mRNA.
  NM_139046Homo sapiens mitogen-activated protein kinase 8 (MAPK8), transcript variant 3, mRNA.
  NM_139047Homo sapiens mitogen-activated protein kinase 8 (MAPK8), transcript variant 4, mRNA.
  NM_002752Homo sapiens mitogen-activated protein kinase 9 (MAPK9), transcript variant 1, mRNA.
  NM_139068Homo sapiens mitogen-activated protein kinase 9 (MAPK9), transcript variant 2, mRNA.
  NM_139069Homo sapiens mitogen-activated protein kinase 9 (MAPK9), transcript variant 3, mRNA.
  NM_139070Homo sapiens mitogen-activated protein kinase 9 (MAPK9), transcript variant 4, mRNA.
  NM_002753Homo sapiens mitogen-activated protein kinase 10 (MAPK10), transcript variant 1, mRNA
  NM_138982Homo sapiens mitogen-activated protein kinase 10 (MAPK10), transcript variant 2, mRNA
  NM_138980Homo sapiens mitogen-activated protein kinase 10 (MAPK10), transcript variant 3, mRNA
  NM_138981Homo sapiens mitogen-activated protein kinase 10 (MAPK10), transcript variant 4, mRNA
  NM_002751Homo sapiens mitogen-activated protein kinase 11 (MAPK11), transcript variant 1, mRNA.
  NM_138993Homo sapiens mitogen-activated protein kinase 11 (MAPK11), transcript variant 2, mRNA.
  NM_002969Homo sapiens mitogen-activated protein kinase 12 (MAPK12), mRNA.
  NM_002754Homo sapiens mitogen-activated protein kinase 13 (MAPK13), mRNA.
  NM_001315Homo sapiens mitogen-activated protein kinase 14 (MAPK14), transcript variant 1, mRNA.
  NM_139012Homo sapiens mitogen-activated protein kinase 14 (MAPK14), transcript variant 2, mRNA.
  NM_139013Homo sapiens mitogen-activated protein kinase 14 (MAPK14), transcript variant 3, mRNA.
  NM_139014Homo sapiens mitogen-activated protein kinase 14 (MAPK14), transcript variant 4, mRNA.
  NM_002755Homo sapiens mitogen-activated protein kinase kinase 1 (MAP2K1), mRNA
  NM_030662Homo sapiens mitogen-activated protein kinase kinase 2 (MAP2K2), mRNA
  NM_002756Homo sapiens mitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant A, mRNA
  NM_145109Homo sapiens mitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant B, mRNA
  NM_145110Homo sapiens mitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant C, mRNA
  XM_008654Homo sapiens mitogen-activated protein kinase kinase 4 (MAP2K4), mRNA
  NM_003010Homo sapiens mitogen-activated protein kinase kinase 4 (MAP2K4), mRNA
  NM_145160Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant A, mRNA
  NM_002757Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant B, mRNA
  NM_145161Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant C, mRNA
  NM_145162Homo sapiens mitogen-activated protein kinase kinase 5 (MAP2K5), transcript variant D, mRNA
  XM_113313Homo sapiens mitogen-activated protein kinase kinase 6 (MAP2K6), mRNA
  NM_002758Homo sapiens mitogen-activated protein kinase kinase 6 (MAP2K6), transcript variant 1, mRNA
  NM_031988Homo sapiens mitogen-activated protein kinase kinase 6 (MAP2K6), transcript variant 2, mRNA
  NM_005043Homo sapiens mitogen-activated protein kinase kinase 7 (MAP2K7), transcript variant A, mRNA
  NM_145185Homo sapiens mitogen-activated protein kinase kinase 7 (MAP2K7), transcript variant B, mRNA
  NM_145329Homo sapiens mitogen-activated protein kinase kinase 7 (MAP2K7), transcript variant C, mRNA
  AF042838Homo sapiens mitogen-activated protein kinase kinase kinase 1 (MAP3K1), mRNA
  NM_006609Homo sapiens mitogen-activated protein kinase kinase kinase 2 (MAP3K2), mRNA
  NM_002401Homo sapiens mitogen-activated protein kinase kinase kinase 3 (MAP3K3), mRNA
  NM_005922Homo sapiens mitogen-activated protein kinase kinase kinase 4 (MAP3K4), transcript variant 1, mRNA
  NM_006724Homo sapiens mitogen-activated protein kinase kinase kinase 4 (MAP3K4), transcript variant 2, mRNA
  NM_005923Homo sapiens mitogen-activated protein kinase kinase kinase 5 (MAP3K5), mRNA
  NM_004672Homo sapiens mitogen-activated protein kinase kinase kinase 6 (MAP3K6), mRNA
  NM_003188Homo sapiens mitogen-activated protein kinase kinase kinase 7 (MAP3K7), mRNA
  NM_005204Homo sapiens mitogen-activated protein kinase kinase kinase 8 (MAP3K8), mRNA
  AF251442Homo sapiens mitogen-activated protein kinase kinase kinase 9 (MAP3K9), mRNA
  NM_002446Homo sapiens mitogen-activated protein kinase kinase kinase 10 (MAP3K10), mRNA
  NM_002419Homo sapiens mitogen-activated protein kinase kinase kinase 11 (MAP3K11), mRNA
  NM_006301Homo sapiens mitogen-activated protein kinase kinase kinase 12 (MAP3K12), mRNA
  NM_004721Homo sapiens mitogen-activated protein kinase kinase kinase 13 (MAP3K13), mRNA
  NM_003954Homo sapiens mitogen-activated protein kinase kinase kinase 14 (MAP3K14), mRNA
  NM_007181Homo sapiens mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), mRNA
  NM_004579Homo sapiens mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), mRNA
  NM_003618Homo sapiens mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3), mRNA
  NM_004834Homo sapiens mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), mRNA
  NM_006575Homo sapiens mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5), mRNA
  NM_003668Homo sapiens mitogen-activated protein kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 1, mRNA
  NM_139078Homo sapiens mitogen-activated protein kinase-activated protein kinase 5 (MAPKAPK5), transcript variant 2, mRNA
  NM_004635Homo sapiens mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3), mRNA
  NM_004759Homo sapiens mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), transcript variant 1, mRNA
  NM_032960Homo sapiens mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), transcript variant 2, mRNA
  NM_005373Homo sapiens myeloproliferative leukemia virus oncogene (MPL), mRNA
  NM_016848Homo sapiens neuronal Shc (SHC3), mRNA
  NM_002649Homo sapiens phosphoinositide-3-kinase, catalytic, gamma polypeptide (PIK3CG), mRNA
  NM_021003Homo sapiens protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform (PPM1A), mRNA
  NM_003942Homo sapiens ribosomal protein S6 kinase, 90 kD, polypeptide 4 (RPS6KA4), mRNA
  NM_004755Homo sapiens ribosomal protein S6 kinase, 90 kD, polypeptide 5 (RPS6KA5), mRNA
  NM_002228Homo sapiens v-jun sarcoma virus 17 oncogene homolog (avian) (JUN), mRNA
 
[00002] [TABLE-US-00002]
  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