RNA Interference Mediated Inhibition Of Interleukin and Interleukin Gene Expression Using Short Interfering Nucleic Acid (siNA)

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27) and/or interleukin receptors (e.g., IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26, and IL-27R) gene expression and/or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in interleukin and/or interleukin receptor (IL and/or IL-R) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. 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 or that mediate RNA interference (RNAi) against interleukin and/or interleukin receptor, such as interleukin-4 and/or interleukin-4 receptor or interleukin-13 and/or interleukin-13 receptor gene expression. Such small nucleic acid molecules are useful, for example, in providing compositions for treatment of traits, diseases and conditions that can respond to modulation of interleukin and/or interleukin receptor expression in a subject, such as cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, disorders, or conditions.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). The corresponding process in plants (Heifetz et al., International PCT Publication No. WO 99/61631) is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fingi. 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 through a mechanism that has yet to be fully characterized. This mechanism appears to be different from other known mechanisms involving double stranded RNA-specific ribonucleases, such as 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 (see for example U.S. Pat. Nos. 6,107,094; 5,898,031; Clemens et al., 1997, J Interferon & Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293). Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; 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 (Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., International PCT Publication No. WO 01/75164, 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 and Tuschl et al., International PCT Publication No. WO 01/75164) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy(2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the guide sequence (Elbashir et al, 2001, EMBO J, 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al, 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164). 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′-O-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 dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, 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.

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 long (141 bp-488 bp) enzymatically synthesized or vector expressed dsRNAs for attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describes certain methods for inhibiting the expression of particular genes in mammalian cells using certain long (550 bp-714 bp), enzymatically synthesized or vector expressed dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for introducing certain long dsRNA molecules into cells for use in inhibiting gene expression in nematodes. 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 long dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Pachuck et al., International PCT Publication No. WO 00/63364, describe certain long (at least 200 nucleotides) dsRNA constructs. 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 and 1998, PNAS, 95, 13959-13964, 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 expression constructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describe specific chemically-modified dsRNA 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 describes 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 long (over 250 bp), vector expressed 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 describe certain methods for inhibiting gene expression using dsRNA. 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 (299 bp-1033 bp) constructs that mediate RNAi. Martinez et al., 2002, Cell, 110, 563-574, describe certain single stranded siRNA constructs, including certain 5′-phosphorylated single stranded siRNAs that mediate RNA interference in Hela cells. Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105, describe certain chemically and structurally modified siRNA molecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically and structurally modified siRNA molecules. Woolf et al., International PCT Publication Nos. WO 03/064626 and WO 03/064625 describe certain chemically modified dsRNA constructs. Hornung et al., 2005, Nature Medicine, 11, 263-270, describe the sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Judge et al., 2005, Nature Biotechnology, Published online: 20 Mar. 2005, describe the sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Yuki et al., International PCT Publication Nos. WO 05/049821 and WO 04/048566, describe certain methods for designing short interfering RNA sequences and certain short interfering RNA sequences with optimized activity. Saigo et al., US Patent Application Publication No. US20040539332, describe certain methods of designing oligo- or polynucleotide sequences, including short interfering RNA sequences, for achieving RNA interference. Tei et al., International PCT Publication No. WO 03/044188, describe certain methods for inhibiting expression of a target gene, which comprises transfecting a cell, tissue, or individual organism with a double-stranded polynucleotide comprising DNA and RNA having a substantially identical nucleotide sequence with at least a partial nucleotide sequence of the target gene.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods useful for modulating interleukins (e.g., IL-1-IL-27) and/or interleukin receptor (e.g., IL-1R-IL-27R) gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of interleukin and/or interleukin receptor gene expression and/or activity by RNA interference (RNAi) using small nucleic acid 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 interleukin and/or interleukin receptor (e.g., IL-1-IL-27 and/or IL-1R-IL-27R) 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 interleukin and/or interleukin receptor 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, cosmetic, veterinary, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of interleukin and/or interleukin receptor genes encoding proteins, such as proteins comprising interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27) and/or interleukin receptors (e.g., IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26, and IL-27R) associated with the maintenance and/or development of cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, traits, conditions and disorders, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I and U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536, both incorporated by reference herein referred to herein generally as interleukin and/or interleukin receptor. The description below of the various aspects and embodiments of the invention is provided with reference to exemplary interleukin and/or interleukin receptor genes referred to herein as interleukin and/or interleukin receptor. However, the various aspects and embodiments are also directed to other interleukin and/or interleukin receptor genes, such as homolog genes and transcript variants, and polymorphisms (e.g., single nucleotide polymorphism, (SNPs)) associated with certain interleukin and/or interleukin receptor genes. As such, the various aspects and embodiments are also directed to other genes that are involved in interleukin and/or interleukin receptor mediated pathways of signal transduction or gene expression that are involved, for example, in the maintenance or development of diseases, traits, conditions, or disorders described herein. These additional genes can be analyzed for target sites using the methods described for interleukin and/or interleukin receptor genes herein. Thus, the modulation of other genes and the effects of such modulation of the other genes can be performed, determined, and measured as described herein.

In one embodiment, the invention features a double stranded nucleic acid molecule, such as a siNA molecule, where one of the strands comprises nucleotide sequence having complementarity to a predetermined interleukin and/or interleukin receptor sequence in a interleukin and/or interleukin receptor target nucleic acid molecule, or a portion thereof. In one embodiment, the predetermined interleukin and/or interleukin receptor nucleotide sequence is a interleukin and/or interleukin receptor nucleotide target sequence described herein. In another embodiment, the predetermined interleukin and/or interleukin receptor sequence is a interleukin and/or interleukin receptor target sequence as is known in the art.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of an interleukin and/or interleukin receptor RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 28 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the interleukin and/or interleukin receptor RNA for the siNA molecule to direct cleavage of the interleukin and/or interleukin receptor RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of an interleukin and/or interleukin receptor RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 23 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the interleukin and/or interleukin receptor RNA for the siNA molecule to direct cleavage of the interleukin and/or interleukin receptor RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of an interleukin and/or interleukin receptor RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 28 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the interleukin and/or interleukin receptor RNA for the siNA molecule to direct cleavage of the interleukin and/or interleukin receptor RNA via RNA interference.

In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of an interleukin and/or interleukin receptor RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 23 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the interleukin and/or interleukin receptor RNA for the siNA molecule to direct cleavage of the interleukin and/or interleukin receptor RNA via RNA interference.

In one embodiment, the invention features a siNA molecule that down-regulates expression of an interleukin and/or interleukin receptor gene or that directs cleavage of a interleukin and/or interleukin receptor RNA, for example, wherein the interleukin and/or interleukin receptor gene or RNA comprises interleukin and/or interleukin receptor encoding sequence. In one embodiment, the invention features a siNA molecule that down-regulates expression of a interleukin and/or interleukin receptor gene or that directs cleavage of a interleukin and/or interleukin receptor RNA, for example, wherein the interleukin and/or interleukin receptor gene or RNA comprises interleukin and/or interleukin receptor non-coding sequence or regulatory elements involved in interleukin and/or interleukin receptor gene expression (e.g., non-coding RNA).

In one embodiment, a siNA of the invention is used to inhibit the expression of interleukin and/or interleukin receptor genes or an interleukin and/or interleukin receptor gene family (e.g., interleukin and/or interleukin receptor superfamily genes), wherein the genes or gene family sequences share sequence homology. Such homologous sequences can be identified as is known in the art, for example using sequence alignments. siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. In instances where mismatches are identified, non-canonical base pairs (for example, mismatches and/or wobble bases) can be used to generate siNA molecules that target more than one gene sequence. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing interleukin and/or interleukin receptor targets that share sequence homology. As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the homologous genes. In this approach, a single siNA can be used to inhibit expression of more than one gene instead of using more than one siNA molecule to target the different genes.

In one embodiment, the invention features a siNA molecule having RNAi activity against interleukin and/or interleukin receptor RNA (e.g., coding or non-coding RNA), wherein the siNA molecule comprises a sequence complementary to any RNA having interleukin and/or interleukin receptor encoding sequence, such as those sequences having GenBank Accession Nos. shown in Table I and U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536, both incorporated by reference herein. In another embodiment, the invention features a siNA molecule having RNAi activity against interleukin and/or interleukin receptor RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having variant interleukin and/or interleukin receptor encoding sequence, for example other mutant interleukin and/or interleukin receptor genes not shown in Table I but known in the art to be associated with the maintenance and/or development of cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, disorders, and/or conditions described herein or otherwise known in the art that are associated with interleukin and/or interleukin gene expression or activity. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention. In another embodiment, a siNA molecule of the invention includes a nucleotide sequence that can interact with nucleotide sequence of an interleukin and/or interleukin receptor gene and thereby mediate silencing of interleukin and/or interleukin receptor gene expression, for example, wherein the siNA mediates regulation of interleukin and/or interleukin receptor gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the interleukin and/or interleukin receptor gene and prevent transcription of the interleukin and/or interleukin receptor gene.

In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of proteins arising from interleukin and/or interleukin receptor haplotype polymorphisms that are associated with a trait, disease or condition (e.g., cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, disorders, and/or conditions). Analysis of genes, or protein or RNA levels can be used to identify subjects with such polymorphisms or those subjects who are at risk of developing traits, conditions, or diseases described herein (see for example Lin et al, 2003, New Engl. J. Med., 349, 2201-2210; Witkin et al., 2002, Clin Infect Dis., 34(2), 204-9; and Keen, 2002, ASHI Quarterly, 4, 152). These subjects are amenable to treatment, for example, treatment with siNA molecules of the invention and any other composition useful in treating diseases related to interleukin and/or interleukin receptor gene expression. As such, analysis of interleukin and/or interleukin receptor protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of interleukin and/or interleukin receptor protein or RNA levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of certain interleukin and/or interleukin receptor proteins associated with a trait, disorder, condition, or disease.

In one embodiment of the invention a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding an interleukin and/or interleukin receptor protein. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of an interleukin and/or interleukin receptor gene or a portion thereof.

In another embodiment, a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a interleukin and/or interleukin receptor protein or a portion thereof. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of an interleukin and/or interleukin receptor gene or a portion thereof.

In one embodiment, the sense region or sense strand of a siNA molecule of the invention is complementary to that portion of the antisense region or antisense strand of the siNA molecule that is complementary to a target polynucleotide sequence.

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 an interleukin and/or interleukin receptor 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 an interleukin and/or interleukin receptor gene sequence or a portion thereof.

In one embodiment, the antisense region of siNA constructs comprises a sequence complementary to sequence having any of target SEQ ID NOs. shown in Tables II and III. In one embodiment, the antisense region of siNA constructs of the invention constructs comprises sequence having any of antisense (lower) SEQ ID NOs. in Tables II and III and FIGS. 4 and 5. In another embodiment, the sense region of siNA constructs of the invention comprises sequence having any of sense (upper) SEQ ID NOs. in Tables II and III and FIGS. 4 and 5.

In one embodiment, a siNA molecule of the invention comprises any of SEQ ID NOs. 1-1260 and 1269-2358. The sequences shown in SEQ ID NOs: 1-1260 and 1269-2358 are not limiting. A siNA molecule of the invention can comprise any contiguous interleukin and/or interleukin receptor sequence (e.g., about 15 to about 25 or more, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous interleukin and/or interleukin receptor nucleotides).

In yet another embodiment, the invention features a siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I and U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536, both incorporated by reference herein. Chemical modifications in Tables III and IV and otherwise described herein can be applied to any siNA construct of the invention.

In one embodiment of the invention a siNA molecule comprises an antisense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is complementary to a RNA sequence or a portion thereof encoding interleukin and/or interleukin receptor, and wherein said siNA further comprises a sense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences where at least about 15 nucleotides in each strand are complementary to the other strand.

In another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region is complementary to a RNA sequence encoding interleukin and/or interleukin receptor, and wherein said siNA further comprises a sense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein said sense region and said antisense region are comprised in a linear molecule where the sense region comprises at least about 15 nucleotides that are complementary to the antisense region.

In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by one or more interleukin and/or interleukin receptor genes. Because interleukin and/or interleukin receptor (e.g., interleukin and/or interleukin receptor superfamily) genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of interleukin and/or interleukin receptor genes or alternately specific interleukin and/or interleukin receptor genes (e.g., polymorphic variants) by selecting sequences that are either shared amongst different interleukin and/or interleukin receptor targets or alternatively that are unique for a specific interleukin and/or interleukin receptor target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of interleukin and/or interleukin receptor RNA sequences having homology among several interleukin and/or interleukin receptor gene variants so as to target a class of interleukin and/or interleukin receptor genes with one siNA molecule. Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of one or both interleukin and/or interleukin receptor alleles in a subject. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific interleukin and/or interleukin receptor RNA sequence (e.g., a single interleukin and/or interleukin receptor allele or interleukin and/or interleukin receptor single nucleotide polymorphism (SNP)) due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, a siNA of the invention is used to inhibit the expression of interleukin and/or interleukin receptor genes, wherein the interleukin and/or interleukin receptor sequences share sequence homology. Such homologous sequences can be identified as is known in the art, for example using sequence alignments. siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. In instances where mismatches are shown, non-canonical base pairs, for example mismatches and/or wobble bases, can be used to generate siNA molecules that target one or more interleukin and/or interleukin receptor RNA sequences. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting differing interleukin and/or interleukin receptor sequences. As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the interleukin and/or interleukin receptor sequences such that the siNA can interact with RNAs of interleukin and/or interleukin receptor and mediate RNAi to achieve inhibition of expression of the interleukin and/or interleukin receptor sequences. In this approach, a single siNA can be used to inhibit expression of more than one interleukin and/or interleukin receptor sequence instead of using more than one siNA molecule to target the different sequences.

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 duplex nucleic acid molecules containing about 15 to about 30 base pairs between oligonucleotides comprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21-nucleotide duplexes with about 19 base pairs and 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with blunt ends, where both ends are blunt, or alternatively, where one of the ends is blunt.

In one embodiment, the nucleotides comprising the overhang portion of a siNA molecule of the invention are complementary to the target polynucleotide sequence and are optionally chemically modified as described herein. In one embodiment, the overhang comprises a 3′-GC or 3′-UU overhang that is complementary to a portion of the target polynucleotide sequence. In another embodiment, the nucleotides comprising the overhanging portion of a siNA molecule of the invention are 2′-O-methyl nucleotides and/or 2′-deoxy-2′-fluoro nucleotides.

In one embodiment, the nucleotides comprising the overhang portion of a siNA molecule of the invention are not complementary to the target polynucleotide sequence and are optionally chemically modified as described herein. In one embodiment, the overhang comprises a 3′-GC or 3′-UU overhang that is not complementary to a portion of the target polynucleotide sequence. In another embodiment, the nucleotides comprising the overhanging portion of a siNA molecule of the invention are 2′-O-methyl nucleotides and/or 2′-deoxy-2′-fluoro nucleotides.

In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for interleukin and/or interleukin receptor expressing nucleic acid molecules, such as RNA encoding an interleukin and/or interleukin receptor protein or non-coding RNA associated with the expression of interleukin and/or interleukin receptor genes. In one embodiment, the invention features a RNA based siNA molecule (e.g., a siNA comprising 2′-OH nucleotides) having specificity for interleukin and/or interleukin receptor expressing nucleic acid molecules that includes one or more chemical modifications described herein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides (see for example U.S. Ser. No. 10/981,966 filed Nov. 5, 2004, incorporated by reference herein), “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, (e.g., RNA based siNA constructs), are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.

In one embodiment, a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, toxicity, immune response, 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., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). For example, in one embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid sugar modification, such as a 2′-sugar modification, e.g., 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-O-methoxyethyl nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, or 2′-deoxy nucleotides. In another embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid base modification, such as 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), or propyne modifications. In another embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid backbone modification, such as a backbone modification having Formula I herein. In another embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid sugar, base, or backbone modification or any combination thereof (e.g., any combination of nucleic acid sugar, base, backbone or non-nucleotide modifications herein). 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.

A siNA molecule of the invention can comprise modified nucleotides at various locations within the siNA molecule. In one embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at internal base paired positions within the siNA duplex. For example, internal positions can comprise positions from about 3 to about 19 nucleotides from the 5′-end of either sense or antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3′-overhangs. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at non-base paired or overhang regions of the siNA molecule. By “non-base paired” is meant, the nucleotides are not base paired between the sense strand or sense region and the antisense strand or antisense region or the siNA molecule. The overhang nucleotides can be complementary or base paired to a corresponding target polynucleotide sequence. For example, overhang positions can comprise positions from about 20 to about 21 nucleotides from the 5′-end of either sense or antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3′-overhangs. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at terminal positions of the siNA molecule. For example, such terminal regions include the 3′-position, 5′-position, for both 3′ and 5′-positions of the sense and/or antisense strand or region of the siNA molecule. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at base-paired or internal positions, non-base paired or overhang regions, and/or terminal regions, or any combination thereof.

One aspect of the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of an interleukin and/or interleukin receptor gene or that directs cleavage of an interleukin and/or interleukin receptor RNA. In one embodiment, the double stranded siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long. In one embodiment, the double-stranded siNA molecule does not contain any ribonucleotides. In another embodiment, the double-stranded siNA molecule comprises one or more ribonucleotides. In one embodiment, each strand of the double-stranded siNA molecule independently comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein each strand comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of the interleukin and/or interleukin receptor gene, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the interleukin and/or interleukin receptor gene or a portion thereof.

In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of an interleukin and/or interleukin receptor gene or that directs cleavage of an interleukin and/or interleukin receptor RNA, comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the interleukin and/or interleukin receptor gene or a portion thereof, and a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the interleukin and/or interleukin receptor gene or a portion thereof. In one embodiment, the antisense region and the sense region independently comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region.

In one embodiment, a siNA molecule of the invention comprises blunt ends, i.e., ends that do not include any overhanging nucleotides. For example, a siNA molecule comprising modifications described herein (e.g., comprising nucleotides having Formulae I-VII or siNA constructs comprising “Stab 00”-“Stab 34” or “Stab 3F”-“Stab 34F” (Table IV) or any combination thereof (see Table IV)) and/or any length described herein can comprise blunt ends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise one or more blunt ends, i.e. where a blunt end does not have any overhanging nucleotides. In one embodiment, the blunt ended siNA molecule has a number of base pairs equal to the number of nucleotides present in each strand of the siNA molecule. In another embodiment, the siNA molecule comprises one blunt end, for example wherein the 5′-end of the antisense strand and the 3′-end of the sense strand do not have any overhanging nucleotides. In another example, the siNA molecule comprises one blunt end, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand do not have any overhanging nucleotides. In another example, a siNA molecule comprises two blunt ends, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand as well as the 5′-end of the antisense strand and 3′-end of the sense strand do not have any overhanging nucleotides. A blunt ended siNA molecule can comprise, for example, from about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). Other nucleotides present in a blunt ended siNA molecule can comprise, for example, mismatches, bulges, loops, or wobble base pairs to modulate the activity of the siNA molecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a double stranded siNA molecule having no overhanging nucleotides. The two strands of a double stranded siNA molecule align with each other without over-hanging nucleotides at the termini. For example, a blunt ended siNA construct comprises terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises ribonucleotides at positions that maintain or enhance RNAi activity. In one embodiment, ribonucleotides are present in the sense strand or sense region of the siNA molecule, which can provide for RNAi activity by allowing cleavage of the sense strand or sense region by RISC (e.g., ribonucleotides present at positions 9 and 10 of the sense strand or sense region). In another embodiment, ribonucleotides are present at 5′-end of the antisense strand or antisense region of the siNA molecule, which can provide for RNAi activity by improving helicase activity or recognition or the siNA by RISC.

In one embodiment, a siNA molecule of the invention contains at least 2, 3, 4, 5, or more chemical modifications that can be the same of different. In another embodiment, a siNA molecule of the invention contains at least 2, 3, 4, 5, or more different chemical modifications.

In one embodiment, a siNA molecule of the invention is a double-stranded short interfering nucleic acid (siNA), wherein the double stranded nucleic acid molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of the nucleotide positions in each strand of the siNA molecule comprises a chemical modification. In one embodiment, the siNA contains at least 2, 3, 4, 5, or more chemical modifications that can be the same of different. In another embodiment, the siNA contains at least 2, 3, 4, 5, or more different chemical modifications.

In one embodiment, the invention features double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of an interleukin and/or interleukin receptor gene or that directs cleavage of an interleukin and/or interleukin receptor RNA, wherein the siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein each strand of the siNA molecule comprises one or more chemical modifications. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of an interleukin and/or interleukin receptor gene or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the interleukin and/or interleukin receptor gene. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of an interleukin and/or interleukin receptor gene or portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or portion thereof of the interleukin and/or interleukin receptor gene. In another embodiment, each strand of the siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and each strand comprises at least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. The interleukin and/or interleukin receptor gene can comprise, for example, sequences referred to in Table I or otherwise described herein or incorporated herein by reference.

In one embodiment, each strand of a double stranded siNA molecule of the invention comprises a different pattern of chemical modifications, such as any “Stab 00”-“Stab 34” or “Stab 3F”-“Stab 34F” (Table IV) modification patterns herein or any combination thereof (see Table IV). Non-limiting examples of sense and antisense strands of such siNA molecules having various modification patterns are shown in Table III.

In one embodiment, a siNA molecule of the invention comprises no ribonucleotides. In another embodiment, a siNA molecule of the invention comprises ribonucleotides.

In one embodiment, a siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of an interleukin and/or interleukin receptor gene or a portion thereof, and the siNA further comprises a sense region comprising a nucleotide sequence substantially similar to the nucleotide sequence of the interleukin and/or interleukin receptor gene or a portion thereof. In another embodiment, the antisense region and the sense region each comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides and the antisense region comprises at least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. The interleukin and/or interleukin receptor gene can comprise, for example, sequences referred to in Table I or incorporated by reference herein. In another embodiment, the siNA is a double stranded nucleic acid molecule, where each of the two strands of the siNA molecule independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides, and where one of the strands of the siNA molecule comprises at least about 15 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more) nucleotides that are complementary to the nucleic acid sequence of the interleukin and/or interleukin receptor gene or a portion thereof.

In one embodiment, a siNA molecule of the invention comprises a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by an interleukin and/or interleukin receptor gene, or a portion thereof, and the sense region comprises a nucleotide sequence that is complementary to the antisense region. In one embodiment, 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. In another embodiment, the sense region is connected to the antisense region via a linker molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule, such as a nucleotide or non-nucleotide linker. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. The interleukin and/or interleukin receptor gene can comprise, for example, sequences referred in to Table I or incorporated by reference herein.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of an interleukin and/or interleukin receptor gene or that directs cleavage of an interleukin and/or interleukin receptor RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the interleukin and/or interleukin receptor gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and 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 one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. 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, 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′-deoxy purine nucleotides. In one embodiment, the pyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the sense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a siNA molecule comprising at least one modified nucleotide, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide, 2′-O-trifluoromethyl nucleotide, 2′-O-ethyl-trifluoromethoxy nucleotide, or 2′-O-difluoromethoxy-ethoxy nucleotide or any other modified nucleoside/nucleotide described herein and in U.S. Ser. No. 10/981,966 filed Nov. 5, 2004, incorporated by reference herein. In one embodiment, the invention features a siNA molecule comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) modified nucleotides, wherein the modified nucleotide is selected from the group consisting of 2′-deoxy-2′-fluoro nucleotide, 2′-O-trifluoromethyl nucleotide, 2′-O-ethyl-trifluoromethoxy nucleotide, or 2′-O-difluoromethoxy-ethoxy nucleotide or any other modified nucleoside/nucleotide described herein and in U.S. Ser. No. 10/981,966, filed Nov. 5, 2004, incorporated by reference herein. The modified nucleotide/nucleoside can be the same or different. The siNA can be, for example, about 15 to about 40 nucleotides in length. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy, 4′-thio pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing the stability of a siNA molecule against cleavage by ribonucleases comprising introducing at least one modified nucleotide into the siNA molecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides.

In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as a phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the antisense region of a siNA molecule of the invention comprises sequence complementary to a portion of an endogenous transcript having sequence unique to a particular interleukin and/or interleukin receptor disease or trait related allele in a subject or organism, such as sequence comprising a single nucleotide polymorphism (SNP) associated with the disease or trait specific allele. As such, the antisense region of a siNA molecule of the invention can comprise sequence complementary to sequences that are unique to a particular allele to provide specificity in mediating selective RNAi against the disease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of an interleukin and/or interleukin receptor gene or that directs cleavage of an interleukin and/or interleukin receptor RNA, 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. In one embodiment, each strand of the double stranded siNA molecule, is about 21 nucleotides long where 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, 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 another embodiment, the siNA molecule is a double stranded nucleic acid molecule, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In another embodiment, all 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, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the interleukin and/or interleukin receptor gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the interleukin and/or interleukin receptor gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of an interleukin and/or interleukin receptor RNA sequence (e.g., wherein said target RNA sequence is encoded by an interleukin and/or interleukin receptor gene involved in the interleukin and/or interleukin receptor pathway), wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the double-stranded siNA molecule is about 15 to about 30 nucleotides. In one embodiment, the siNA molecule is 21 nucleotides in length. Examples of non-ribonucleotide containing siNA constructs are combinations of stabilization chemistries shown in Table IV in any combination of Sense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense or antisense strands or any combination thereof). Herein, numeric Stab chemistries can include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table IV. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc. In one embodiment, the invention features a chemically synthesized double stranded RNA molecule that directs cleavage of an interleukin and/or interleukin receptor RNA via RNA interference, wherein each strand of said RNA molecule is about 15 to about 30 nucleotides in length; one strand of the RNA molecule comprises nucleotide sequence having sufficient complementarity to the interleukin and/or interleukin receptor RNA for the RNA molecule to direct cleavage of the interleukin and/or interleukin receptor RNA via RNA interference; and wherein at least one strand of the RNA molecule optionally comprises one or more chemically modified nucleotides described herein, such as without limitation deoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-O-methoxyethyl nucleotides, 4′-thio nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, etc.

In one embodiment, an interleukin and/or interleukin receptor RNA of the invention comprises sequence encoding an interleukin and/or interleukin receptor protein.

In one embodiment, an interleukin and/or interleukin receptor RNA of the invention comprises non-coding RNA sequence (e.g., miRNA, snRNA, siRNA etc.), see for example Mattick, 2005, Science, 309, 1527-1528 and Clayerie, 2005, Science, 309, 1529-1530.

In one embodiment, the invention features a medicament comprising a siNA molecule of the invention.

In one embodiment, the invention features an active ingredient comprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to inhibit, down-regulate, or reduce expression of a interleukin and/or interleukin receptor gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is independently about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more) nucleotides long. In one embodiment, the siNA molecule of the invention is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each of the two fragments of the siNA molecule independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and where one of the strands comprises at least 15 nucleotides that are complementary to nucleotide sequence of interleukin and/or interleukin receptor encoding RNA or a portion thereof. In a non-limiting example, each of the two fragments of the siNA molecule comprise about 21 nucleotides. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 21 nucleotide long and where 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, 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 another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In another embodiment, all 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, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region and comprising one or more chemical modifications, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the interleukin and/or interleukin receptor gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the interleukin and/or interleukin receptor gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor 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. In one embodiment, each strand has at least two (e.g., 2, 3, 4, 5, or more) chemical modifications, which can be the same or different, such as nucleotide, sugar, base, or backbone modifications. In one embodiment, a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, a majority of the purine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor RNA that encodes a protein or 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, each strand of the siNA molecule comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides, wherein each strand comprises at least about 15 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 one 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 a further 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 still another 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′-deoxy purine nucleotides. In another embodiment, the antisense strand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methyl purine nucleotides. In another 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 a further embodiment the sense strand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety (e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotide moiety such as inverted thymidine) is present at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand. In another 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 any of the above-described embodiments of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of an interleukin and/or interleukin receptor gene, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, each of the two strands of the siNA molecule can comprise about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule. In another embodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule, wherein 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 another 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 one 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 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of the antisense strand are base-paired to the nucleotide sequence of the interleukin and/or interleukin receptor RNA or a portion thereof. In one embodiment, about 18 to about 25 (e.g., about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisense strand are base-paired to the nucleotide sequence of the interleukin and/or interleukin receptor RNA or a portion thereof.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor 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. In one embodiment, each strand has at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, such as nucleotide sugar, base, or backbone modifications. In one embodiment, a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, a majority of the purine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one 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 inhibits expression of an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor 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 or a portion thereof of the antisense strand is complementary to a nucleotide sequence of the untranslated region or a portion thereof of the interleukin and/or interleukin receptor RNA.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand, 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 interleukin and/or interleukin receptor RNA or a portion thereof that is present in the interleukin and/or interleukin receptor RNA.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention in a pharmaceutically acceptable carrier or diluent.

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 or immunostimulation in humans.

In any of the embodiments of siNA molecules described herein, the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.

One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to a RNA or DNA sequence encoding interleukin and/or interleukin receptor 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.

wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified and which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule, 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, aralkyl, or acetyl and wherein W, X, Y, and Z are optionally not all O. In another embodiment, a backbone modification of the invention comprises a phosphonoacetate and/or thiophosphonoacetate internucleotide linkage (see for example Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

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.

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-OSH, 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 any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; 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. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties 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; steroids, and polyamines, such as PEI, spermine or spermidine.

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 nucleotides or non-nucleotides 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.

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-OSH, 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 any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; 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. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties 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; steroids, and polyamines, such as PEI, spermine or spermidine.

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 nucleotides or non-nucleotides of Formula III at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

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, alkylhalo, or acetyl; and wherein W, X, Y and Z are optionally not all 0 and Y serves as a point of attachment to the siNA molecule.

In one embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand, for example, a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminal nucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target-complementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor 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.

Each strand of the double stranded siNA molecule can have one or more chemical modifications such that each strand comprises a different pattern of chemical modifications. Several non-limiting examples of modification schemes that could give rise to different patterns of modifications are provided herein (see for example Stab chemistries shown in Table IV, and double stranded nucleic acid molecules having any of SI, SII, SIII, SIV, SV, and/or SVI).

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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein the antisense 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio 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, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5 or more (specifically about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one or both siNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand is independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex has about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, 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 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, 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 to about 21 (e.g., 19, 20, or 21) base pairs and a 2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises a hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications 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 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). 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. In one embodiment, a linear hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises an asymmetric hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications 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 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms an asymmetric hairpin structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In one embodiment, an asymmetric hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In another embodiment, an asymmetric hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, wherein the sense region and the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications 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 an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) nucleotides in length and wherein the sense region is about 3 to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides in length, wherein the sense region the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. In another embodiment, the asymmetric double stranded siNA molecule can also have a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV).

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 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF₃, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, 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 any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties 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; steroids, and polyamines, such as PEI, spermine or spermidine.

In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, 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 any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siNA molecule of the invention. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties 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; steroids, and polyamines, such as PEI, spermine or spermidine.

In another embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula VII:

wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, 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 any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule. In one embodiment, R3 and/or R1 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties 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; steroids, and polyamines, such as PEI, spermine or spermidine.

By “ZIP code” sequences is meant, any peptide or protein sequence that is involved in cellular topogenic signaling mediated transport (see for example Ray et al., 2004, Science, 306(1501): 1505)

Each nucleotide within the double stranded siNA molecule can independently have a chemical modification comprising the structure of any of Formulae I-VIII. Thus, in one embodiment, one or more nucleotide positions of a siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein. In one embodiment, each nucleotide position of a siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein.

In one embodiment, one or more nucleotide positions of one or both strands of a double stranded siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein. In one embodiment, each nucleotide position of one or both strands of a double stranded siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein.

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).

In another embodiment, a chemically modified nucleoside or non-nucleoside (e.g. 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, chemically modified nucleoside or non-nucleoside (e.g., 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 one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the terminal position of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the two terminal positions of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the penultimate position of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In addition, a moiety having Formula VII can be present at the 3′-end or the 5′-end of a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula VI or VI is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In 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) 4′-thio nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein 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, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule, of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor inside a cell or reconstituted in vitro system comprising a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and 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 an antisense region, wherein one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or more purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). The sense region and/or the antisense region can have 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 sense and/or antisense sequence. The sense and/or antisense region can optionally further comprise a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhang nucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein. In any of these described embodiments, the purine nucleotides present in the sense region are alternatively 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides) and one or more purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Also, in any of these embodiments, one or more purine nucleotides present in the sense region are alternatively purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides) and any purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Additionally, in any of these embodiments, one or more purine nucleotides present in the sense region and/or present in the antisense region are alternatively selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides 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, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides 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, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides).

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) otherwise known as a “ribo-like” or “A-form helix” configuration. 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, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, 4′-thio nucleotides and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA molecule of the invention comprises a terminal cap moiety, (see for example FIG. 10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. Non-limiting examples of conjugates contemplated by the invention include conjugates and ligands described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein in its entirety, including the drawings. 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 cholesterol, polyethylene glycol, human serum albumin, or a ligand for a cellular receptor, 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; steroids, and polyamines, such as PEI, spermine or spermidine. 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, filed Jul. 22, 2002 incorporated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker is used, for example, to attach a conjugate moiety to the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of >2 nucleotides in length, for example about 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; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 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.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides. For example, a siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA comprise separate oligonucleotides that do not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotides. In another example, a siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotide. Applicant has surprisingly found that the presence of ribonucleotides (e.g., nucleotides having a 2′-hydroxyl group) within the siNA molecule is not required or essential to support RNAi activity. As such, in one embodiment, all positions within the siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy 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 comprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more) 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, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group. In any of these embodiments, any purine nucleotides present in the antisense region are alternatively 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). Also, in any of these embodiments, any purine nucleotides present in the siNA (i.e., purine nucleotides present in the sense and/or antisense region) can alternatively be locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA are alternatively 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). In another embodiment, any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.

In one embodiment, a siNA molecule of the invention comprises chemically modified nucleotides or non-nucleotides (e.g., having any of Formulae I-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides) at alternating positions within one or more strands or regions of the siNA molecule. For example, such chemical modifications can be introduced at every other position of a RNA based siNA molecule, starting at either the first or second nucleotide from the 3′-end or 5′-end of the siNA. In a non-limiting example, a double stranded siNA molecule of the invention in which each strand of the siNA is 21 nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., with compounds having any of Formulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In another non-limiting example, a double stranded siNA molecule of the invention in which each strand of the siNA is 21 nucleotides in length is featured wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand are chemically modified (e.g., with compounds having any of Formulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In one embodiment, one strand of the double stranded siNA molecule comprises chemical modifications at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 and chemical modifications at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21. Such siNA molecules can further comprise terminal cap moieties and/or backbone modifications as described herein.

In one embodiment, a siNA molecule of the invention comprises the following features: if purine nucleotides are present at the 5′-end (e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 from the 5′-end) of the antisense strand or antisense region (otherwise referred to as the guide sequence or guide strand) of the siNA molecule then such purine nucleosides are ribonucleotides. In another embodiment, the purine ribonucleotides, when present, are base paired to nucleotides of the sense strand or sense region (otherwise referred to as the passenger strand) of the siNA molecule. Such purine ribonucleotides can be present in a siNA stabilization motif that otherwise comprises modified nucleotides.

In one embodiment, a siNA molecule of the invention comprises the following features: if pyrimidine nucleotides are present at the 5′-end (e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 from the 5′-end) of the antisense strand or antisense region (otherwise referred to as the guide sequence or guide strand) of the siNA molecule then such pyrimidine nucleosides are ribonucleotides. In another embodiment, the pyrimidine ribonucleotides, when present, are base paired to nucleotides of the sense strand or sense region (otherwise referred to as the passenger strand) of the siNA molecule. Such pyrimidine ribonucleotides can be present in a siNA stabilization motif that otherwise comprises modified nucleotides.

In one embodiment, a siNA molecule of the invention comprises the following features: if pyrimidine nucleotides are present at the 5′-end (e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 from the 5′-end) of the antisense strand or antisense region (otherwise referred to as the guide sequence or guide strand) of the siNA molecule then such pyrimidine nucleosides are modified nucleotides. In another embodiment, the modified pyrimidine nucleotides, when present, are base paired to nucleotides of the sense strand or sense region (otherwise referred to as the passenger strand) of the siNA molecule. Non-limiting examples of modified pyrimidine nucleotides include those having any of Formulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides.

In one embodiment, the invention features a double stranded nucleic acid molecule having structure SI:

wherein each N is independently a nucleotide; each B is a terminal cap moiety that can be present or absent; (N) represents non-base paired or overhanging nucleotides which can be unmodified or chemically modified; [N] represents nucleotide positions wherein any purine nucleotides when present are ribonucleotides; X1 and X2 are independently integers from about 0 to about 4; X3 is an integer from about 9 to about 21; X4 is an integer from about 11 to about 20, provided that the sum of X4 and X5 is between 17-21; X5 is an integer from about 1 to about 6; and

(a) any pyrimidine nucleotides present in the antisense strand (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the antisense strand (lower strand) other than the purines nucleotides in the [N] nucleotide positions, are independently 2′-O-methyl nucleotides, 2′-deoxyribonucleotides or a combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides;

(b) any pyrimidine nucleotides present in the sense strand (upper strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the sense strand (upper strand) are independently 2′-deoxyribonucleotides, 2′-O-methyl nucleotides or a combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides; and

In one embodiment, the invention features a double stranded nucleic acid molecule having structure SII:

(b) any pyrimidine nucleotides present in the sense strand (upper strand) are ribonucleotides; any purine nucleotides present in the sense strand (upper strand) are ribonucleotides; and

In one embodiment, the invention features a double stranded nucleic acid molecule having structure SIII:

(a) any pyrimidine nucleotides present in the antisense strand (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the antisense strand

(lower strand) other than the purines nucleotides in the [N] nucleotide positions, are 2′-O-methyl nucleotides;

In one embodiment, the invention features a double stranded nucleic acid molecule having structure SIV:

In one embodiment, the invention features a double stranded nucleic acid molecule having structure SV:

(a) any pyrimidine nucleotides present in the antisense strand (lower strand) are nucleotides having a ribo-like configuration (e.g., Northern or A-form helix configuration); any purine nucleotides present in the antisense strand (lower strand) other than the purines nucleotides in the [N] nucleotide positions, are 2′-O-methyl nucleotides;

(b) any pyrimidine nucleotides present in the sense strand (upper strand) are nucleotides having a ribo-like configuration (e.g., Northern or A-form helix configuration); any purine nucleotides present in the sense strand (upper strand) are 2′-O-methyl nucleotides; and

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, or SVI comprises an antisense strand having complementarity to a Interleukin and/or interleukin receptor target polynucleotide (e.g., Interleukin and/or interleukin receptor RNA or DNA). In another embodiment, the Interleukin and/or interleukin receptor target polynucleotide is DSG1, DSG2, DSG3, and/or DSG4 RNA and/or DNA. In another embodiment, the Interleukin and/or interleukin receptor target polynucleotide is conserved across all Interleukin and/or interleukin receptor isoforms.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, or SVI comprises a terminal phosphate group at the 5′-end of the antisense strand or antisense region of the nucleic acid molecule.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, or SVI comprises X5=1, 2, or 3; each X1 and X2=1 or 2; X3=12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and X4=15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, or SVI comprises X5=1; each X1 and X2=2; X3=19, and X4=18.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, or SVI comprises X5=2; each X1 and X2=2; X3=19, and X4=17

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, or SVI comprises X5=3; each X1 and X2=2; X3=19, and X4=16.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, or SVI comprises B at the 3′ and 5′ ends of the sense strand or sense region.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, or SVI comprises B at the 3′-end of the antisense strand or antisense region.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SI, SIII, SIV, SV, or SVI comprises B at the 3′ and 5′ ends of the sense strand or sense region and B at the 3′-end of the antisense strand or antisense region.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, or SVI further comprises one or more phosphorothioate internucleotide linkages at the first terminal (N) on the 3′ end of the sense strand, antisense strand, or both sense strand and antisense strands of the nucleic acid molecule. For example, a double stranded nucleic acid molecule can comprise X1 and/or X2=2 having overhanging nucleotide positions with a phosphorothioate internucleotide linkage, e.g., (NsN) where “s” indicates phosphorothioate.

In one embodiment, the invention features a method for modulating the expression of an interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in the cell.

In one embodiment, the invention features a method for modulating the expression of an interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the cell.

In another embodiment, the invention features a method for modulating the expression of two or more interleukin and/or interleukin receptor genes within a cell comprising: (a) synthesizing one or more siNA molecules of the invention, which can be chemically-modified or unmodified, wherein the siNA strands comprise sequences complementary to RNA of the interleukin and/or interleukin receptor genes and wherein the sense strand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the cell.

In another embodiment, the invention features a method for modulating the expression of an interleukin gene and its corresponding receptor gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin gene and the corresponding receptor gene, wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the cell.

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

In one embodiment, the invention features a method of modulating the expression of an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor 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 (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in that organism.

In one embodiment, the invention features a method of modulating the expression of an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar 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 (e.g., inhibit) the expression of the interleukin and/or interleukin receptor 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 (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor 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 (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in that organism.

In one embodiment, the invention features a method of modulating the expression of an interleukin and/or interleukin receptor gene in a subject or 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 interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in the subject or organism. The level of interleukin and/or interleukin receptor protein or RNA can be determined using various methods well-known in the art.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor gene in a subject or 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 interleukin and/or interleukin receptor genes; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the subject or organism. The level of interleukin and/or interleukin receptor protein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating the expression of an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor gene; and (b) contacting the cell in vitro or in vivo with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the cell.

In one embodiment, the invention features a method of modulating the expression of an interleukin and/or interleukin receptor gene in a tissue explant (e.g., a skin, heart, liver, spleen, cornea, lung, stomach, kidney, vein, artery, hair, appendage, or limb transplant, or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) 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 interleukin and/or interleukin receptor gene; and (b) contacting a cell of the tissue explant derived from a particular subject or organism with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in that subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor gene in a tissue explant (e.g., a skin, heart, liver, spleen, cornea, lung, stomach, kidney, vein, artery, hair, appendage, or limb transplant, or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) 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 interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in that subject or organism.

In one embodiment, the invention features a method of modulating the expression of an interleukin and/or interleukin receptor gene in a subject or 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 interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in the subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor gene in a subject or 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 interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the subject or organism.

In one embodiment, the invention features a method of modulating the expression of an interleukin and/or interleukin receptor gene in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor gene in the subject or organism.

In one embodiment, the invention features a method for treating or preventing an inflammatory, disease, disorder, or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the subject or organism whereby the treatment or prevention of inflammatory, disease, disorder, and/or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as tissues or cells affected by the inflammatory disease, disorder, or condition. Non-limiting examples of such tissues include lung, sinus, or nasopharyngeal tissues and cells, such as airway epithelial cells; gastrointestinal tissues and cells; CNS or PNS tissues and cells; cardiovascular tissues and cells; dermal or subcutaneous tissues and cells; liver tissues and cells; kidney tissues and cells, bladder tissues and cells; colorectal tissues and cells; synovial tissues and cells; musculoskeletal tissues and cells; ocular tissues and cells; lymphatic tissues and cells such as T-cells, B-cells, or macrophages; hematopoetic tissues and cells etc. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells affected by the inflammatory disease, disorder, or condition. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing a respiratory, disease, disorder, and/or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the subject or organism whereby the treatment or prevention of respiratory, disease, disorder, and/or condition can be achieved. In one embodiment, the interleukin or interleukin receptor gene is IL-4, IL-4R, IL-5, IL-5R, IL-7, IL-7R, IL-9, IL-9R, IL-13, or IL-13R. In one embodiment, the respiratory disease is asthma, COPD, allergic rhinitis, or any other reparatory disease herein or otherwise known in the art (see for example Corry et al., 2002, Am. J. Resp. Med., 1, 185-193 and Blease et al., 2003, Exp. Opinion Emerging Drugs, 8, 71-81). In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as tissues or cells affected by the respiratory disease, disorder, or condition. Non-limiting examples of such tissues include lung, sinus, or nasopharyngeal tissues and cells, such as airway epithelial cells, mast cells, alveolar cells, bronchial epithelial cells, bronchial smooth muscle cells, and normal human lung fibroblasts. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells affected by the respiratory disease, disorder, or condition. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for inhibiting or reducing airway hyperresponsiveness in a subject or organism, comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of an appropriate interleukin and/or appropriate interleukin receptor gene in the subject or organism whereby the inhibition or reduction in the airway hyperresponsiveness can be achieved. In one embodiment, the interleukin or interleukin receptor gene is IL-4, IL-4R, IL-5, IL-5R, IL-7, IL-7R, IL-9, IL-9R, IL-13, or IL-13R. In one embodiment, the airway hyperresponsiveness is associated with asthma, COPD, allergic rhinitis, or any other reparatory disease herein or otherwise known in the art (see for example Corry et al., 2002, Am. J. Resp. Med., 1, 185-193 and Blease et al., 2003, Exp. Opinion Emerging Drugs, 8, 71-81). In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as tissues or cells affected by the respiratory disease, disorder, or condition. Non-limiting examples of such tissues include lung, sinus, or nasopharyngeal tissues and cells, such as airway epithelial cells, mast cells, alveolar cells, bronchial epithelial cells, bronchial smooth muscle cells, and normal human lung fibroblasts. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells affected by the airway hyperresponsiveness. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing a autoimmune disease, disorder, and/or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the subject or organism whereby the treatment or prevention of autoimmune, disease, disorder, and/or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as tissues or cells affected by the autoimmune disease, disorder, or condition. Non-limiting examples of such tissues include lung, sinus, or nasopharyngeal tissues and cells, such as airway epithelial cells; gastrointestinal tissues and cells; CNS or PNS tissues and cells; cardiovascular tissues and cells; dermal or subcutaneous tissues and cells; liver tissues and cells; kidney tissues and cells, bladder tissues and cells; colorectal tissues and cells; synovial tissues and cells; musculoskeletal tissues and cells; ocular tissues and cells; lymphatic tissues and cells such as T-cells, B-cells, or macrophages; hematopoetic tissues and cells etc. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells affected by the autoimmune disease, disorder, or condition. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing a cardiovascular disease, disorder, and/or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the subject or organism whereby the treatment or prevention of cardiovascular, disease, disorder, and/or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as tissues or cells affected by the cardiovascular disease, disorder, or condition. Non-limiting examples of such tissues and cells include vascular epithelial tissues and cells and/or cardiac tissues and cells etc. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells affected by the cardiovascular disease, disorder, or condition. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing a neurological disease, disorder, and/or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the subject or organism whereby the treatment or prevention of neurological, disease, disorder, and/or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as tissues or cells affected by the neurological disease, disorder, or condition. Non-limiting examples of such tissues include CNS (e.g., brain and spinal cord) or PNS tissues and cells such as glial cells, neurons, astrocytes, microglia, dendrites, etc. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells affected by the neurological disease, disorder, or condition. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing a proliferative disease, disorder, and/or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the subject or organism whereby the treatment or prevention of proliferative, disease, disorder, and/or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as tissues or cells affected by the proliferative disease, disorder, or condition. Non-limiting examples of such tissues include lung, sinus, or nasopharyngeal tissues and cells, such as airway epithelial cells; gastrointestinal tissues and cells; CNS or PNS tissues and cells; cardiovascular tissues and cells; dermal or subcutaneous tissues and cells; liver tissues and cells; kidney tissues and cells, bladder tissues and cells; colorectal tissues and cells; synovial tissues and cells; musculoskeletal tissues and cells; ocular tissues and cells; lymphatic tissues and cells such as T-cells, B-cells, or macrophages; hematopoetic tissues and cells etc. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells affected by the proliferative disease, disorder, or condition. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing cancer in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the subject or organism whereby the treatment or prevention of cancer can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as tissues or cells affected by the cancer. Non-limiting examples of such tissues include lung, sinus, or nasopharyngeal tissues and cells, such as airway epithelial cells; gastrointestinal tissues and cells; CNS or PNS tissues and cells; cardiovascular tissues and cells; dermal or subcutaneous tissues and cells; liver tissues and cells; kidney tissues and cells, bladder tissues and cells; colorectal tissues and cells; synovial tissues and cells; musculoskeletal tissues and cells; ocular tissues and cells; lymphatic tissues and cells such as T-cells, B-cells, or macrophages; hematopoetic tissues and cells etc. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells affected by the cancer. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor gene in a subject or organism comprising contacting the subject or organism with one or more siNA molecules of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the subject or organism.

In one embodiment, the invention features a method of modulating the expression of a interleukin and/or interleukin receptor target gene in a tissue explant (e.g., skin, hair, lung, or any other tissue or cell as can be transplanted from one organism to another or back to the same organism from which the tissue or cell is derived) 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 interleukin and/or interleukin receptor target gene; and (b) contacting a cell of the tissue explant derived from a particular subject or organism with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor target gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor target gene in that subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor target gene in a tissue explant (e.g., skin, hair, lung, or any other tissue or cell as can be transplanted from one organism to another or back to the same organism from which the tissue or cell is derived) 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 interleukin and/or interleukin receptor target gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor target genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor target genes in that subject or organism.

In one embodiment, the invention features a method for treating or preventing a disease, disorder, trait or condition related to gene expression in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor target gene in the subject or organism. The reduction of gene expression and thus reduction in the level of the respective protein/RNA relieves, to some extent, the symptoms of the disease, disorder, trait or condition.

In one embodiment, the invention features a method for treating or preventing a dermatological disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor target gene in the subject or organism whereby the treatment or prevention of the dermatological disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the dermatological disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the dermatological disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to interleukin and/or interleukin receptor target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of dermatological diseases, traits, disorders, or conditions in a subject or organism.

In any of the methods of treatment of the invention, the siNA can be administered to the subject as a course of treatment, for example administration at various time intervals, such as once per day over the course of treatment, once every two days over the course of treatment, once every three days over the course of treatment, once every four days over the course of treatment, once every five days over the course of treatment, once every six days over the course of treatment, once per week over the course of treatment, once every other week over the course of treatment, once per month over the course of treatment, etc. In one embodiment, the course of treatment is from about one to about 52 weeks or longer (e.g., indefinitely). In one embodiment, the course of treatment is from about one to about 48 months or longer (e.g., indefinitely).

In any of the methods of treatment of the invention, the siNA can be administered to the subject systemically as described herein or otherwise known in the art. Systemic administration can include, for example, intravenous, subcutaneous, intramuscular, catheterization, nasopharyngeal, transdermal, or gastrointestinal administration as is generally known in the art.

In any of the methods of treatment of the invention, the siNA can be administered to the subject locally or to local tissues as described herein, either alone as a monotherapy or in combination with additional therapies as are known in the art. Local administration can include, for example, intraocular, periocular, nasopharyngeal, inhalation, nebulization, implantation, dermal/transdermal application, or direct injection to relevant tissues, or any other local administration technique, method or procedure, as is generally known in the art.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin or interleukin receptor gene in a subject or organism comprising contacting the subject or organism with one or more siNA molecules of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the interleukin and/or interleukin receptor genes in the subject or organism.

The siNA molecules of the invention can be designed to down regulate or inhibit target (e.g., interleukin and/or interleukin receptor) gene expression through RNAi targeting of a variety of nucleic acid molecules. In one embodiment, the siNA molecules of the invention are used to target various DNA corresponding to a target gene, for example via heterochromatic silencing or transcriptional inhibition. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene, for example via RNA target cleavage or translational inhibition. Non-limiting examples of such RNAs include messenger RNA (mRNA), non-coding RNA (ncRNA) or regulatory elements (see for example Mattick, 2005, Science, 309, 1527-1528 and Clayerie, 2005, Science, 309, 1529-1530) which includes miRNA and other small RNAs, 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, cosmetic applications, veterinary applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as interleukin and/or interleukin receptor gene families having homologous sequences. As such, siNA molecules targeting multiple interleukin and/or interleukin receptor genes or RNA targets can provide increased therapeutic effect. In one embodiment, the invention features the targeting (cleavage or inhibition of expression or function) of more than one IL or IL-R gene sequence using a single siNA molecule, by targeting the conserved sequences of the targeted IL or IL-R gene.

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, inflammatory, respiratory, autoimmune, neurological, cardiovascular, and/or proliferative diseases, traits, and conditions associated with interleukin and/or interleukin receptor gene expression or activity in a subject or organism.

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, interleukin and/or interleukin receptor genes encoding RNA sequence(s) referred to herein by GenBank Accession number, for example, GenBank Accession Nos. shown in Table I, U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536 as incorporated by reference herein.

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 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4^N, where N represents the number of base paired nucleotides in each of the siNA construct strands (e.g. 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 interleukin and/or interleukin receptor 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 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 6 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor RNA sequence. The target interleukin and/or interleukin receptor RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In one embodiment, the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In another embodiment, the siNA molecules of (b) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for diagnosing a disease, trait, or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease, trait, or condition in the subject. In another embodiment, the invention features a method for treating or preventing a disease, trait, or condition (e.g., cancer, inflammatory, respiratory, autoimmune, neurological, cardiovascular, and/or proliferative diseases, traits, or conditions) in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease, trait, or condition in the subject, alone or in conjunction with one or more other therapeutic compounds.

In another embodiment, the invention features a method for validating an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor target gene; (b) introducing the siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modulating expression of the interleukin and/or interleukin receptor target gene in the cell, tissue, subject, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, subject, or organism.

In another embodiment, the invention features a method for validating an interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the interleukin and/or interleukin receptor target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human or animal, wherein the system comprises the components required for RNAi activity. The term “biological system” includes, for example, a cell, tissue, subject, or organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.

By “phenotypic change” is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA). Such detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of an interleukin and/or interleukin receptor target gene in a biological system, including, for example, in a cell, tissue, subject, 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 interleukin and/or interleukin receptor target gene in a biological system, including, for example, in a cell, tissue, subject, or organism.

In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified. In another embodiment, the cell containing a siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention, which can be chemically-modified, comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions using an alkylamine base such as methylamine. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In one embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.

In another embodiment, the invention features a method for making a double-stranded siNA molecule in a single synthetic process comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein 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.

In another embodiment, the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein in their entirety.

In one embodiment, the invention features siNA constructs that mediate RNAi against interleukin and/or interleukin receptor target polynucleotide (e.g., interleukin and/or interleukin RNA or DNA), wherein the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.

In another embodiment, the invention features a method for generating siNA molecules with improved toxicologic profiles (e.g., having attenuated or no immunstimulatory properties) comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) 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 toxicologic profiles.

In another embodiment, the invention features a method for generating siNA formulations with improved toxicologic profiles (e.g., having attenuated or no immunstimulatory properties) comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations having improved toxicologic profiles.

In another embodiment, the invention features a method for generating siNA molecules that do not stimulate an interferon response (e.g., no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate an interferon response.

In another embodiment, the invention features a method for generating siNA formulations that do not stimulate an interferon response (e.g., no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations that do not stimulate an interferon response. In one embodiment, the interferon comprises interferon alpha.

In another embodiment, the invention features a method for generating siNA molecules that do not stimulate an inflammatory or proinflammatory cytokine response (e.g., no cytokine response or attenuated cytokine response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table I) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate a cytokine response. In one embodiment, the cytokine comprises an interleukin such as interleukin-6 (IL-6) and/or tumor necrosis alpha (TNF-a).

In another embodiment, the invention features a method for generating siNA formulations that do not stimulate an inflammatory or proinflammatory cytokine response (e.g., no cytokine response or attenuated cytokine response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations that do not stimulate a cytokine response. In one embodiment, the cytokine comprises an interleukin such as interleukin-6 (IL-6) and/or tumor necrosis alpha (TNF-a).

In another embodiment, the invention features a method for generating siNA molecules that do not stimulate Toll-like Receptor (TLR) response (e.g., no TLR response or attenuated TLR response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table I) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate a TLR response. In one embodiment, the TLR comprises TLR3, TLR7, TLR8 and/or TLR9.

In another embodiment, the invention features a method for generating siNA formulations that do not stimulate a Toll-like Receptor (TLR) response (e.g., no TLR response or attenuated TLR response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations that do not stimulate a TLR response. In one embodiment, the TLR comprises TLR3, TLR7, TLR8 and/or TLR9.

In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein: (a) each strand of said siNA molecule is about 18 to about 38 nucleotides in length; (b) one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference; and (c) wherein the nucleotide positions within said siNA molecule are chemically modified to reduce the immunstimulatory properties of the siNA molecule to a level below that of a corresponding unmodified siRNA molecule. Such siNA molecules are said to have an improved toxicologic profile compared to an unmodified or minimally modified siNA.

By “improved toxicologic profile”, is meant that the chemically modified or formulated siNA construct exhibits decreased toxicity in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology. In a non-limiting example, siNA molecules and formulations with improved toxicologic profiles are associated with reduced immunostimulatory properties, such as a reduced, decreased or attenuated immunostimulatory response in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology. Such an improved toxicologic profile is characterized by abrogated or reduced immunostimulation, such as reduction or abrogation of induction of interferons (e.g., interferon alpha), inflammatory cytokines (e.g., interleukins such as IL-6, and/or TNF-alpha), and/or toll like receptors (e.g., TLR-3, TLR-7, TLR-8, and/or TLR-9). In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises no ribonucleotides. In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32, Stab 33, Stab 34 or any combination thereof (see Table IV). In one embodiment, the level of immunostimulatory response associated with a given siNA molecule can be measured as is known in the art, for example by determining the level of PKR/interferon response, proliferation, B-cell activation, and/or cytokine production in assays to quantitate the immunostimulatory response of particular siNA molecules (see, for example, Leifer et al., 2003, J Immunother. 26, 313-9; and U.S. Pat. No. 5,968,909, incorporated in its entirety by reference). Herein, numeric Stab chemistries include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table IV. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc. In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises a siNA molecule of the invention and a formulation as described in United States Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety including the drawings.

In one embodiment, the level of immunostimulatory response associated with a given siNA molecule can be measured as is described herein or as is otherwise known in the art, for example by determining the level of PKR/interferon response, proliferation, B-cell activation, and/or cytokine production in assays to quantitate the immunostimulatory response of particular siNA molecules (see, for example, Leifer et al., 2003, J Immunother. 26, 313-9; and U.S. Pat. No. 5,968,909, incorporated in its entirety by reference). In one embodiment, the reduced immunostimulatory response is between about 10% and about 100% compared to an unmodified or minimally modified siRNA molecule, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduced immunostimulatory response. In one embodiment, the immunostimulatory response associated with a siNA molecule can be modulated by the degree of chemical modification. For example, a siNA molecule having between about 10% and about 100%, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the nucleotide positions in the siNA molecule modified can be selected to have a corresponding degree of immunostimulatory properties as described herein.

In one embodiment, the invention features a chemically synthesized double stranded siNA molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein (a) each strand of said siNA molecule is about 18 to about 38 nucleotides in length; (b) one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference; and (c) wherein one or more nucleotides of said siNA molecule are chemically modified to reduce the immunostimulatory properties of the siNA molecule to a level below that of a corresponding unmodified siNA molecule. In one embodiment, each strand comprises at least about 18 nucleotides that are complementary to the nucleotides of the other strand.

In another embodiment, the siNA molecule comprising modified nucleotides to reduce the immunostimulatory properties of the siNA molecule comprises an antisense region having nucleotide sequence that is complementary to a nucleotide sequence of a target gene or a portion thereof and further comprises a sense region, wherein said sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of said target gene or portion thereof. In one embodiment thereof, the antisense region and the sense region comprise about 18 to about 38 nucleotides, wherein said antisense region comprises at least about 18 nucleotides that are complementary to nucleotides of the sense region. In one embodiment thereof, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides. In another embodiment thereof, the purine nucleotides in the sense region are 2′-deoxy purine nucleotides. In yet another embodiment thereof, the pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In another embodiment thereof, the pyrimidine nucleotides of said antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In yet another embodiment thereof, the purine nucleotides of said antisense region are 2′-O-methyl purine nucleotides. In still another embodiment thereof, the purine nucleotides present in said antisense region comprise 2′-deoxypurine nucleotides. In another embodiment, the antisense region comprises a phosphorothioate internucleotide linkage at the 3′ end of said antisense region. In another embodiment, the antisense region comprises a glyceryl modification at a 3′ end of said antisense region.

In other embodiments, the siNA molecule comprising modified nucleotides to reduce the immunostimulatory properties of the siNA molecule can comprise any of the structural features of siNA molecules described herein. In other embodiments, the siNA molecule comprising modified nucleotides to reduce the immunostimulatory properties of the siNA molecule can comprise any of the chemical modifications of siNA molecules described herein.

In one embodiment, the invention features a method for generating a chemically synthesized double stranded siNA molecule having chemically modified nucleotides to reduce the immunostimulatory properties of the siNA molecule, comprising (a) introducing one or more modified nucleotides in the siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating an siNA molecule having reduced immunostimulatory properties compared to a corresponding siNA molecule having unmodified nucleotides. Each strand of the siNA molecule is about 18 to about 38 nucleotides in length. One strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference. In one embodiment, the reduced immunostimulatory properties comprise an abrogated or reduced induction of inflammatory or proinflammatory cytokines, such as interleukin-6 (IL-6) or tumor necrosis alpha (TNF-a), in response to the siNA being introduced in a cell, tissue, or organism. In another embodiment, the reduced immunostimulatory properties comprise an abrogated or reduced induction of Toll Like Receptors (TLRs), such as TLR3, TLR7, TLR8 or TLR9, in response to the siNA being introduced in a cell, tissue, or organism.

In another embodiment, the reduced immunostimulatory properties comprise an abrogated or reduced induction of interferons, such as interferon alpha, in response to the siNA being introduced in a cell, tissue, or organism.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence.

In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.

In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against interleukin and/or interleukin receptor target polynucleotide in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against interleukin and/or interleukin receptor comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity. In another embodiment, the invention features a method for generating siNA molecules with improved RNAi specificity against interleukin and/or interleukin receptor polynucleotide targets 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 specificity. In one embodiment, improved specificity comprises having reduced off target effects compared to an unmodified siNA molecule. For example, introduction of terminal cap moieties at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand or region of a siNA molecule of the invention can direct the siNA to have improved specificity by preventing the sense strand or sense region from acting as a template for RNAi activity against a corresponding target having complementarity to the sense strand or sense region.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor target RNA.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against interleukin and/or interleukin receptor target polynucleotide comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor target RNA.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against interleukin and/or interleukin receptor target DNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target DNA.

In another embodiment, the invention features a method for generating siNA molecules against interleukin and/or interleukin receptor target polynucleotide with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediate RNAi against interleukin and/or interleukin receptor target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing a conjugate into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; cholesterol derivatives, polyamines, such as spermine or spermidine; and others.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is chemically modified in a manner that it can no longer act as a guide sequence for efficiently mediating RNA interference and/or be recognized by cellular proteins that facilitate RNAi. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified (e.g., is all RNA).

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein the second sequence is designed or modified in a manner that prevents its entry into the RNAi pathway as a guide sequence or as a sequence that is complementary to a target nucleic acid (e.g., RNA) sequence. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified (e.g., is all RNA). Such design or modifications are expected to enhance the activity of siNA and/or improve the specificity of siNA molecules of the invention. These modifications are also expected to minimize any off-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end of said second sequence. In one embodiment, the terminal cap moiety comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end and 3′-end of said second sequence. In one embodiment, each terminal cap moiety individually comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid (e.g., a DNA or RNA such as a gene or its corresponding RNA), comprising (a) introducing one or more chemical modifications 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 specificity. In another embodiment, the chemical modification used to improve specificity comprises terminal cap modifications at the 5′-end, 3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal cap modifications can comprise, for example, structures shown in FIG. 10 (e.g. inverted deoxyabasic moieties) or any other chemical modification that renders a portion of the siNA molecule (e.g. the sense strand) incapable of mediating RNA interference against an off target nucleic acid sequence. In a non-limiting example, a siNA molecule is designed such that only the antisense sequence of the siNA molecule can serve as a guide sequence for RISC mediated degradation of a corresponding target RNA sequence. This can be accomplished by rendering the sense sequence of the siNA inactive by introducing chemical modifications to the sense strand that preclude recognition of the sense strand as a guide sequence by RNAi machinery. In one embodiment, such chemical modifications comprise any chemical group at the 5′-end of the sense strand of the siNA, or any other group that serves to render the sense strand inactive as a guide sequence for mediating RNA interference. These modifications, for example, can result in a molecule where the 5′-end of the sense strand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphate group (e.g., phosphate, diphosphate, triphosphate, cyclic phosphate etc.). Non-limiting examples of such siNA constructs are described herein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (see Table IV) wherein the 5′-end and 3′-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group. Herein, numeric Stab chemistries include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table IV. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc.

In one embodiment, the invention features a method for screening siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of unmodified siNA molecules, (b) screening the siNA molecules of step (a) under conditions suitable for isolating siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence, and (c) introducing chemical modifications (e.g. chemical modifications as described herein or as otherwise known in the art) into the active siNA molecules of (b). In one embodiment, the method further comprises re-screening the chemically modified siNA molecules of step (c) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

In one embodiment, the invention features a method for screening chemically modified siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of chemically modified siNA molecules (e.g. siNA molecules as described herein or as otherwise known in the art), and (b) screening the siNA molecules of step (a) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly. The receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercellular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.

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, nanoparticles, receptors, ligands, and others.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing nucleotides having any of Formulae I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 100 to about 50,000 daltons (Da).

The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include a siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, U.S. Pat. No. 6,395,713). The kit can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996). Such a kit can also include instructions to allow a user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemically-modified short interfering nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner. For example the siNA can be a double-stranded nucleic acid 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 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; 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 (e.g., about 15 to about 25 or more nucleotides of the siNA molecule are complementary to the target nucleic acid 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 duplex, asymmetric duplex, hairpin or asymmetric 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 embodiments, 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. Non limiting examples of siNA molecules of the invention are shown in FIGS. 4-6, and Table III herein. Such siNA molecules are distinct from other nucleic acid technologies known in the art that mediate inhibition of gene expression, such as ribozymes, antisense, triplex forming, aptamer, 2,5-A chimera, or decoy oligonucleotides.

By “RNA interference” or “RNAi” is meant a biological process of inhibiting or down regulating gene expression in a cell as is generally known in the art and which is mediated by short interfering nucleic acid molecules, see for example Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; see for example Zamore et al., 2000, Cell, 101, 25-33; 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). 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, transcriptional inhibition, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure or methylation patterns to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). In another non-limiting example, modulation of gene expression by siNA molecules of the invention can result from siNA mediated cleavage of RNA (either coding or non-coding RNA) via RISC, or alternately, translational inhibition as is known in the art. In another embodiment, modulation of gene expression by siNA molecules of the invention can result from transcriptional inhibition (see for example Janowski et al., 2005, Nature Chemical Biology, 1, 216-222).

In one embodiment, a siNA molecule of the invention is a duplex forming oligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al., U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCT Application No. US04/16390, filed May 24, 2004).

In one embodiment, a siNA molecule of the invention is a multifunctional siNA, (see for example FIGS. 16-21 and Jadhav et al., U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and International PCT Application No. US04/16390, filed May 24, 2004). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting, for example, two or more regions of interleukin and/or interleukin receptor RNA (see for example target sequences in Tables II and III). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more interleukin and/or interleukin receptor sequences (e.g., IL4, IL4R, IL13, and/or IL13R) coding or non-coding sequences. In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more interleukin and/or interleukin receptor RNA and one or more CHRM3 coding or non-coding sequences (see for example U.S. Ser. No. 10/919,866, incorporated by reference herein). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more interleukin and/or interleukin receptor RNA and one or more ADAM33 coding or non-coding sequences (see for example U.S. Ser. No. 10/923,329; incorporated by reference herein). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more interleukin and/or interleukin receptor RNA and one or more GPRA/AAA1 coding or non-coding sequences (see for example U.S. Ser. No. 10/923,182; incorporated by reference herein). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more interleukin and/or interleukin receptor RNA and one or more ADORA1 coding or non-coding sequences (see for example U.S. Ser. No. 10/224,005; incorporated by reference herein)

By “asymmetric hairpin” as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprising about 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides, and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also comprise a 5′-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g., about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with post transcriptional silencing, such as RNAi mediated cleavage of a target nucleic acid molecule (e.g. RNA) or inhibition of translation. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with pretranscriptional silencing, such as by alterations in DNA methylation patterns and DNA chromatin structure.

By up-regulate”, or “promote”, 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 increased above that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, up-regulation or promotion of gene expression with an siNA molecule is above that level observed in the presence of an inactive or attenuated molecule. In another embodiment, up-regulation or promotion of gene expression with siNA molecules is above that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, up-regulation or promotion of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence. In one embodiment, up-regulation or promotion of gene expression is associated with inhibition of RNA mediated gene silencing, such as RNAi mediated cleavage or silencing of a coding or non-coding RNA target that down regulates, inhibits, or silences the expression of the gene of interest to be up-regulated. The down regulation of gene expression can, for example, be induced by a coding RNA or its encoded protein, such as through negative feedback or antagonistic effects. The down regulation of gene expression can, for example, be induced by a non-coding RNA having regulatory control over a gene of interest, for example by silencing expression of the gene via translational inhibition, chromatin structure, methylation, RISC mediated RNA cleavage, or translational inhibition. As such, inhibition or down regulation of targets that down regulate, suppress, or silence a gene of interest can be used to up-regulate or promote expression of the gene of interest toward therapeutic use.

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

By “non-canonical base pair” is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N-3-amino(H2), GA sheared, UC 4-carbonylamino, WU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, WU 2-carbonyl-imino symmetric, WU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, WU imino-4-carbonyl, AC C2-H—N3, GA carbonyl-C2-H, WU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “interleukin” is meant, any interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27) protein, peptide, or polypeptide having any interleukin activity, such as encoded by interleukin Genbank Accession Nos. shown in Table I. The term interleukin also refers to nucleic acid sequences encoding any interleukin protein, peptide, or polypeptide having interleukin activity. The term “interleukin” is also meant to include other interleukin encoding sequence, such as other interleukin isoforms, mutant interleukin genes, splice variants of interleukin genes, and interleukin gene polymorphisms.

By “interleukin receptor” as used herein is meant, any interleukin receptor (e.g., IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26R, and IL-27R) protein, peptide, or polypeptide having any interleukin receptor activity, such as encoded by interleukin receptor GenBank Accession Nos. shown in Table I and/or in U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536, both incorporated by reference herein. The term interleukin receptor also refers to nucleic acid sequences encoding any interleukin receptor protein, peptide, or polypeptide having interleukin receptor activity. The term “interleukin receptor” is also meant to include other interleukin receptor encoding sequence, such as other interleukin receptor isoforms, mutant interleukin receptor genes, splice variants of interleukin receptor genes, and interleukin receptor gene polymorphisms.

By “corresponding” interleukin receptor is meant, any interleukin receptor that binds to a given interleukin. For example, the corresponding interleukin receptors for IL-4 are IL-4R and IL-13R, as IL-4 is a ligand for both IL-4R and IL-13R.

By “target” as used herein is meant, any target protein, peptide, or polypeptide (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27), such as encoded by GenBank Accession Nos. shown in Table I and/or in U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536, both incorporated by reference herein. The term “target” also refers to nucleic acid sequences or target polynucleotide sequence encoding any target protein, peptide, or polypeptide, such as proteins, peptides, or polypeptides (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27) encoded by sequences having GenBank Accession Nos. shown in Table I and/or in U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536. The target of interest can include target polynucleotide sequences, such as target DNA or target RNA. The term “target” is also meant to include other sequences, such as differing isoforms, mutant target genes, splice variants of target polynucleotides, target polymorphisms, and non-coding (e.g., ncRNA, miRNA, sRNA) or other regulatory polynucleotide sequences as described herein. Therefore, in various embodiments of the invention, a double stranded nucleic acid molecule of the invention (e.g., siNA) having complementarity to a target RNA can be used to inhibit or down regulate miRNA or other ncRNA activity. In one embodiment, inhibition of miRNA or ncRNA activity can be used to down regulate or inhibit gene expression (e.g., gene targets described herein or otherwise known in the art) or viral replication (e.g., viral targets described herein or otherwise known in the art) that is dependent on miRNA or ncRNA activity. In another embodiment, inhibition of miRNA or ncRNA activity by double stranded nucleic acid molecules of the invention (e.g. siNA) having complementarity to the miRNA or ncRNA can be used to up regulate or promote target gene expression (e.g., gene targets described herein or otherwise known in the art) where the expression of such genes is down regulated, suppressed, or silenced by the miRNA or ncRNA. Such up-regulation of gene expression can be used to treat diseases and conditions associated with a loss of function or haploinsufficiency as are generally known in the art.

By “homologous sequence” is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

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. In one embodiment, the sense region of the siNA molecule is referred to as the sense strand or passenger strand

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. In one embodiment, the antisense region of the siNA molecule is referred to as the antisense strand or guide strand.

By “target nucleic acid” or “target polynucleotide” is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA. In one embodiment, a target nucleic acid of the invention is interleukin and/or interleukin receptor RNA or DNA.

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 as described herein. In one embodiment, a double stranded nucleic acid molecule of the invention, such as an siNA molecule, wherein each strand is between 15 and 30 nucleotides in length, comprises between about 10% and about 100% (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the two strands of the double stranded nucleic acid molecule. In another embodiment, a double stranded nucleic acid molecule of the invention, such as an siNA molecule, where one strand is the sense strand and the other stand is the antisense strand, wherein each strand is between 15 and 30 nucleotides in length, comprises between at least about 10% and about 100% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the nucleotide sequence in the antisense strand of the double stranded nucleic acid molecule and the nucleotide sequence of its corresponding target nucleic acid molecule, such as a target RNA or target mRNA or viral RNA. In one embodiment, a double stranded nucleic acid molecule of the invention, such as an siNA molecule, where one strand comprises nucleotide sequence that is referred to as the sense region and the other strand comprises a nucleotide sequence that is referred to as the antisense region, wherein each strand is between 15 and 30 nucleotides in length, comprises between about 10% and about 100% (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the sense region and the antisense region of the double stranded nucleic acid molecule. 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, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). In one embodiment, a siNA molecule of the invention has perfect complementarity between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule. In one embodiment, a siNA molecule of the invention is perfectly complementary to a corresponding target nucleic acid molecule. “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. In one embodiment, a siNA molecule of the invention comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof. In one embodiment, a siNA molecule of the invention has partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule or between the antisense strand or antisense region of the siNA molecule and a corresponding target nucleic acid molecule. For example, partial complementarity can include various mismatches or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides) within the siNA structure which can result in bulges, loops, or overhangs that result between the between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule or between the antisense strand or antisense region of the siNA molecule and a corresponding target nucleic acid molecule.

In one embodiment, a double stranded nucleic acid molecule of the invention, such as siNA molecule, has perfect complementarity between the sense strand or sense region and the antisense strand or antisense region of the nucleic acid molecule. In one embodiment, double stranded nucleic acid molecule of the invention, such as siNA molecule, is perfectly complementary to a corresponding target nucleic acid molecule.

In one embodiment, double stranded nucleic acid molecule of the invention, such as siNA molecule, has partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the double stranded nucleic acid molecule or between the antisense strand or antisense region of the nucleic acid molecule and a corresponding target nucleic acid molecule. For example, partial complementarity can include various mismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides, such as nucleotide bulges) within the double stranded nucleic acid molecule, structure which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the double stranded nucleic acid molecule or between the antisense strand or antisense region of the double stranded nucleic acid molecule and a corresponding target nucleic acid molecule.

In one embodiment, double stranded nucleic acid molecule of the invention is a microRNA (miRNA). By “mircoRNA” or “miRNA” is meant, a small double stranded RNA that regulates the expression of target messenger RNAs either by mRNA cleavage; translational repression/inhibition or heterochromatic silencing (see for example Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531; and Ying et al., 2004, Gene, 342, 25-28). In one embodiment, the microRNA of the invention, has partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the miRNA molecule or between the antisense strand or antisense region of the miRNA and a corresponding target nucleic acid molecule. For example, partial complementarity can include various mismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides, such as nucleotide bulges) within the double stranded nucleic acid molecule, structure which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the miRNA or between the antisense strand or antisense region of the miRNA and a corresponding target nucleic acid molecule.

In one embodiment, siNA molecules of the invention that down regulate or reduce interleukin and/or interleukin receptor gene expression are used for preventing or treating cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, disorders, conditions, or traits in a subject or organism as described herein or otherwise known in the art.

In one embodiment, the siNA molecules of the invention are used to treat cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, disorders, and/or conditions in a subject or organism.

By “proliferative disease” or “cancer” as used herein is meant, any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including leukemias, for example, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS related cancers such as Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers; cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, cancers of the retina such as retinoblastoma, cancers of the esophagus, gastric cancers, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, colorectal cancer, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrug resistant cancers; and proliferative diseases and conditions, such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet/dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration and other proliferative diseases and conditions such as restenosis and polycystic kidney disease, and any other cancer or proliferative disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.

By “inflammatory disease” or “inflammatory condition” as used herein is meant any disease, condition, trait, genotype or phenotype characterized by an inflammatory or allergic process as is known in the art, such as inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowl disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familial eosinophilia (FE), autosomal recessive spastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chronic cholecystitis, Bronchiectasis, Silicosis and other pneumoconiosis, and any other inflammatory disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.

By “autoimmune disease” or “autoimmune condition” as used herein is meant, any disease, condition, trait, genotype or phenotype characterized by autoimmunity as is known in the art, such as multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, and any other autoimmune disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.

By “neurologic disease” or “neurological disease” is meant any disease, disorder, or condition affecting the central or peripheral nervous system, including ADHD, AIDS-Neurological Complications, Absence of the Septum Pellucidum, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Aspartame, Asperger Syndrome, Ataxia Telangiectasia, Ataxia, Attention Deficit-Hyperactivity Disorder, Autism, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain Aneurysm, Brain Injury, Brain and Spinal Tumors, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Cephalic Disorders, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysm, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome, Charcot-Marie-Tooth Disorder, Chiari Malformation, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Coma, including Persistent Vegetative State, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease (CIBD), Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia—Multi-Infarct, Dementia—Subcortical, Dementia With Lewy Bodies, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet's Syndrome, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis Lethargica, Encephalitis and Meningitis, Encephaloceles, Encephalopathy, Encephalotrigeminal Angiomatosis, Epilepsy, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Fabry's Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Spastic Paralysis, Febrile Seizures (e.g., GEFS and GEFS plus), Fisher Syndrome, Floppy Infant Syndrome, Friedreich's Ataxia, Gaucher's Disease, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Guillain-Barre Syndrome, HTLV-1 Associated Myelopathy, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster Oticus, Herpes Zoster, Hirayama Syndrome, Holoprosencephaly, Huntington's Disease, Hydranencephaly, Hydrocephalus—Normal Pressure, Hydrocephalus, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Keine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Klüver-Bucy Syndrome, Korsakoffs Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus—Neurological Sequelae, Lyme Disease—Neurological Complications, Machado-Joseph Disease, Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini-Strokes, Mitochondrial Myopathies, Mobius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy with Orthostatic. Hypotension, Multiple System Atrophy, Muscular Dystrophy, Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus, Myopathy—Congenital, Myopathy—Thyrotoxic, Myopathy, Myotonia Congenita, Myotonia, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Manifestations of Pompe Disease, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathy—Hereditary, Neurosarcoidosis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Occult Spinal Dysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain—Chronic, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Parmyotonia Congenita, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick's Disease, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Lateral Sclerosis, Prion Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Pseudotumor Cerebri, Pyridoxine Dependent and Pyridoxine Responsive Siezure Disorders, Ramsay Hunt Syndrome Type I, Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and other autoimmune epilepsies, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease—Infantile, Refsum Disease, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness, Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Atrophy, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen Disease, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Aftack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis including Temporal Arteritis, Von Economo's Disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease, X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger Syndrome.

By “respiratory disease” is meant, any disease or condition affecting the respiratory tract, such as asthma, chronic obstructive pulmonary disease or “COPD”, bronchiectasis, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergies, impeded respiration, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, Hantavirus pulmonary syndrome (HPS), Loeffler's syndrome, Goodpasture's syndrome, Pleurisy, pneumonitis, pulmonary edema, pulmonary fibrosis, Sarcoidosis, complications associated with respiratory syncitial virus infection, and any other respiratory disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies. Respiratory diseases and conditions are commonly associated with airway hyperresponsiveness mediated by cytokines, including interleukins described herein.

By “airway hyperresponsiveness” as used herein is meant, any disfunction of the respiratory tract that involves increased sensitivity to an airway constrictive or inflammatory agonist, such as environmental allergens. Airway hyperresponsiveness is a characteristic feature of asthma and other respiratory diseases and generally consists of an increased sensitivity of the airways to an inhaled constrictor agonist, a steeper slope of the dose-response curve, and a greater maximal response to the agonist. Measurements of airway responsiveness are useful in making a diagnosis of asthma, particularly in patients who have symptoms that are consistent with asthma and who have no evidence of airflow obstruction. Certain inhaled stimuli, such as environmental allergens, can increase airway inflammation and enhance airway hyperresponsiveness. These changes in airway hyperresponsiveness are of much smaller magnitude than those seen when asthmatic patients with persistent airway hyperresponsiveness are compared to healthy subjects. They are, however, similar to changes occurring in asthmatic patients that are associated with worsening asthma control. The mechanisms of the transient allergen-induced airway hyperresponsiveness are not likely to fully explain the underlying mechanisms of the persistent airway hyperresponsiveness in asthmatic patients (see for example O-Byrne et al., 2003, Chest, 123, 411S-416S).

By “cardiovascular disease” is meant and disease or condition affecting the heart and vasculature, including but not limited to, coronary heart disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular diseases (stroke), transient ischaemic attacks (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, vavular disease, and/or congestive heart failure.

By “dermatological disease” means any disease or condition of the skin, dermis, or any substructure therein such as hair, follicle, etc. Dermatological diseases, disorders, conditions, and traits can include psoriasis, ectopic dermatitis, skin cancers such as melanoma and basal cell carcinoma, hair loss, hair removal, alterations in pigmentation, and any other disease, condition, or trait associated with the skin, dermis, or structures therein.

In one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently about 15 to about 30 nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In another embodiment, one or more strands of the siNA molecule of the invention independently comprises about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) that are complementary to a target nucleic acid molecule. 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., about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15 to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs. Exemplary siNA molecules of the invention are shown in Table II. Exemplary synthetic siNA molecules of the invention are shown in Table III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through local delivery to the lung, direct dermal application, transdermal application, or injection 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.

In another aspect, the invention provides mammalian cells containing one or more siNA molecules of this invention. The one or more siNA molecules can independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

By “chemical modification” as used herein is meant any modification of chemical structure of the nucleotides that differs from nucleotides of native siRNA or RNA. The term “chemical modification” encompasses the addition, substitution, or modification of native siRNA or RNA nucleosides and nucleotides with modified nucleosides and modified nucleotides as described herein or as is otherwise known in the art. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides (see for example U.S. Ser. No. 10/981,966 filed Nov. 5, 2004, incorporated by reference herein), “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, terminal glyceryl and/or inverted deoxy abasic residue incorporation, or a modification having any of Formulae I-VII herein.

The term “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise an acetyl or protected acetyl group.

The term “thiophosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z comprises an acetyl or protected acetyl group and W comprises a sulfur atom or alternately W comprises an acetyl or protected acetyl group and Z comprises a sulfur atom.

The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to for preventing or treating cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, conditions, disorders and traits described herein or otherwise known in the art in a subject or organism.

In one embodiment, a siNA molecule or composition of the invention is used to treat asthma, COPD, allergic rhinitis, emphysema, or any other respiratory disease herein.

In one embodiment, the siNA molecules of the invention can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combination with other known treatments to prevent or treat cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, conditions, disorders and traits described herein in a subject or organism. For example, the described molecules could be used in combination with one or more known compounds, treatments, or procedures to prevent or treat cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, conditions, disorders and traits described herein in a subject or organism as are known in the art.

In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a GenBank Accession numbers, for example GenBank Accession Nos. shown in Table I, U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536, incorporated by reference herein.

In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.

In another aspect of the invention, siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by GenBank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, 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.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.

FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siNA constructs.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety 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”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, 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”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage 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”, optionally 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.

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 IL-13R receptor siNA sequence. Such chemical modifications can be applied to any interleukin and/or interleukin receptor sequence or other target polynucleotide sequence.

FIG. 6A-B shows non-limiting examples of different siNA constructs of the invention. The examples shown in FIG. 6A (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.

The examples shown in FIG. 6B represent different variations of double stranded nucleic acid molecule of the invention, such as microRNA, that can include overhangs, bulges, loops, and stem-loops resulting from partial complementarity. Such motifs having bulges, loops, and stem-loops are generally characteristics of miRNA. The bulges, loops, and stem-loops can result from any degree of partial complementarity, such as mismatches or bulges of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in one or both strands of the double stranded nucleic acid molecule of the invention.

FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined interleukin and/or interleukin receptor target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for a interleukin and/or interleukin receptor target sequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-terminal nucleotide overhang results from the transcription, for example, by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002, Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined interleukin and/or interleukin receptor target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted into vectors such that (FIG. 9C) transfection of a vector into cells results in the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.

FIG. 10 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 3′-end of siNA sequences of the invention, including (1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5) [5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistance while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing 2′-modifications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct in tested in an appropriate system (e.g. human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay). Lead siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are identified in a target nucleic acid sequence. (i) A palindrome or repeat sequence is identified in a nucleic acid target sequence. (ii) A sequence is designed that is complementary to the target nucleic acid sequence and the palindrome sequence. (iii) An inverse repeat sequence of the non-palindrome/repeat portion of the complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO molecule comprising sequence complementary to the nucleic acid target. (iv) The DFO molecule can self-assemble to form a double stranded oligonucleotide. FIG. 14B shows a non-limiting representative example of a duplex forming oligonucleotide sequence. FIG. 14C shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence. FIG. 14D shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence followed by interaction with a target nucleic acid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are incorporated into the DFO constructs that have sequence complementary to any target nucleic acid sequence of interest. Incorporation of these palindrome/repeat sequences allow the design of DFO constructs that form duplexes in which each strand is capable of mediating modulation of target gene expression, for example by RNAi. First, the target sequence is identified. A complementary sequence is then generated in which nucleotide or non-nucleotide modifications (shown as X or Y) are introduced into the complementary sequence that generate an artificial palindrome (shown as XYXYXY in the Figure). An inverse repeat of the non-palindrome/repeat complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO comprising sequence complementary to the nucleic acid target. The DFO can self-assemble to form a double stranded oligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 16A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 16B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 17A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 17B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifunctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 18A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 18B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifunctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 19A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 19B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid molecules, such as separate RNA molecules encoding differing proteins, for example, a cytokine and its corresponding receptor, differing viral strains, a virus and a cellular protein involved in viral infection or replication, or differing proteins involved in a common or divergent biologic pathway that is implicated in the maintenance of progression of disease. Each strand of the multifunctional siNA construct comprises a region having complementarity to separate target nucleic acid molecules. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid sequences within the same target nucleic acid molecule, such as alternate coding regions of a RNA, coding and non-coding regions of a RNA, or alternate splice variant regions of a RNA. Each strand of the multifunctional siNA construct comprises a region having complementarity to the separate regions of the target nucleic acid molecule. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target region. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

FIG. 22(A-H) shows non-limiting examples of tethered multifunctional siNA constructs of the invention. In the examples shown, a linker (e.g., nucleotide or non-nucleotide linker) connects two siNA regions (e.g., two sense, two antisense, or alternately a sense and an antisense region together. Separate sense (or sense and antisense) sequences corresponding to a first target sequence and second target sequence are hybridized to their corresponding sense and/or antisense sequences in the multifunctional siNA. In addition, various conjugates, ligands, aptamers, polymers or reporter molecules can be attached to the linker region for selective or improved delivery and/or pharmacokinetic properties.

FIG. 23 shows a non-limiting example of various dendrimer based multifunctional siNA designs.

FIG. 24 shows a non-limiting example of various supramolecular multifunctional siNA designs.

FIG. 25 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 30 nucleotide precursor siNA construct. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2′-OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30 mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs.

FIG. 26 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 40 nucleotide precursor siNA construct. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Four targeting sequences are shown. The target sequences having homology are enclosed by boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.

FIG. 27 shows a non-limiting example of additional multifunctional siNA construct designs of the invention. In one example, a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.

FIG. 28 shows a non-limiting example of additional multifunctional siNA construct designs of the invention. In one example, a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.

FIG. 29 shows a non-limiting example of IL-4 inhibition in HeLa cells using a dual luciferase reporter system. The IL-4 target site with flanking rat sequences was cloned into the 3′ untranslated region of Renilla luciferase to create a reporter plasmid. Specific siNA-induced degradation of the target sequence in Renilla mRNA transcribed from this plasmid results in a loss of Renilla luciferase signal in plasmid-transfected HeLa cells. The reporter plasmid also contains a copy of the Firefly luciferase gene, which does not contain the target site sequences. In HeLa cells co-transfected with the reporter plasmid and siNAs, the ratio of Renilla to Firefly luciferase activities (using two different substrates) provides a measure of siNA activity. The Firefly luciferase activity provides an internal control for transfection efficiency, toxicity and sample recovery. As shown in the Figure, treatment of the dual luciferase reporter system HeLa cells with 12.5 mM siNA targeting IL-4 resulted in marked inhibition of Renilla luciferase activity after 17 hours compared to untreated cells and cells treated with a matched chemistry inverted control. Compound numbers (see Table III, sense/antisense strand) of the siNA constructs and target sites within the IL-4 target are shown on the X-axis of the plot.

FIG. 30 shows a non-limiting example of IL-13 inhibition in HeLa cells using a dual luciferase reporter system. The IL-13 target site with flanking rat sequences was cloned into the 3′ untranslated region of Renilla luciferase to create a reporter plasmid. Specific siNA-induced degradation of the target sequence in Renilla mRNA transcribed from this plasmid results in a loss of Renilla luciferase signal in plasmid-transfected HeLa cells. The reporter plasmid also contains a copy of the Firefly luciferase gene, which does not contain the target site sequences. In HeLa cells co-transfected with the reporter plasmid and siNAs, the ratio of Renilla to Firefly luciferase activities (using two different substrates) provides a measure of siNA activity. The Firefly luciferase activity provides an internal control for transfection efficiency, toxicity and sample recovery. As shown in the Figure, treatment of the dual luciferase reporter system HeLa cells with 12.5 nM siNA targeting IL-13 resulted in marked inhibition of Renilla luciferase activity after 17 hours compared to untreated cells and cells treated with a matched chemistry inverted control. Compound numbers (see Table III, sense/antisense strand) of the siNA constructs and target sites within the IL-13 target are shown on the X-axis of the plot.

FIG. 31 shows a non-limiting example of a cholesterol linked phosphoramidite that can be used to synthesize cholesterol conjugated siNA molecules of the invention. An example is shown with the cholesterol moiety linked to the 5′-end of the sense strand of a siNA molecule.

DETAILED DESCRIPTION OF THE INVENTION
Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limiting only to siRNA and can be applied to siNA as a whole. By “improved capacity to mediate RNAi” or “improved RNAi activity” is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention. In this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or a siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188). In addition, RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). As such, siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi 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.

Duplex Forming Oligonucleotides (DFO) of the Invention

In one embodiment, the invention features siNA molecules comprising duplex forming oligonucleotides (DFO) that can self-assemble into double stranded oligonucleotides. The duplex forming oligonucleotides of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The DFO molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, referred to herein for convenience but not limitation as duplex forming oligonucleotides or DFO molecules, are potent mediators of sequence specific regulation of gene expression. The oligonucleotides of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides etc.) in that they represent a class of linear polynucleotide sequences that are designed to self-assemble into double stranded oligonucleotides, where each strand in the double stranded oligonucleotides comprises a nucleotide sequence that is complementary to a target nucleic acid molecule. Nucleic acid molecules of the invention can thus self assemble into functional duplexes in which each strand of the duplex comprises the same polynucleotide sequence and each strand comprises a nucleotide sequence that is complementary to a target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotide sequences where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are assembled from two separate oligonucleotides, or from a single molecule that folds on itself to form a double stranded structure, often referred to in the field as hairpin stem-loop structure (e.g., shRNA or short hairpin RNA). These double stranded oligonucleotides known in the art all have a common feature in that each strand of the duplex has a distinct nucleotide sequence.

Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of forming a double stranded nucleic acid molecule starting from a single stranded or linear oligonucleotide. The two strands of the double stranded oligonucleotide formed according to the instant invention have the same nucleotide sequence and are not covalently linked to each other. Such double-stranded oligonucleotides molecules can be readily linked post-synthetically by methods and reagents known in the art and are within the scope of the invention. In one embodiment, the single stranded oligonucleotide of the invention (the duplex forming oligonucleotide) that forms a double stranded oligonucleotide comprises a first region and a second region, where the second region includes a nucleotide sequence that is an inverted repeat of the nucleotide sequence in the first region, or a portion thereof, such that the single stranded oligonucleotide self assembles to form a duplex oligonucleotide in which the nucleotide sequence of one strand of the duplex is the same as the nucleotide sequence of the second strand. Non-limiting examples of such duplex forming oligonucleotides are illustrated in FIGS. 14 and 15. These duplex forming oligonucleotides (DFOs) can optionally include certain palindrome or repeat sequences where such palindrome or repeat sequences are present in between the first region and the second region of the DFO.

In one embodiment, the invention features a duplex forming oligonucleotide (DFO) molecule, wherein the DFO comprises a duplex forming self complementary nucleic acid sequence that has nucleotide sequence complementary to an interleukin and/or interleukin receptor target nucleic acid sequence. The DFO molecule can comprise a single self complementary sequence or a duplex resulting from assembly of such self complementary sequences.

In one embodiment, a duplex forming oligonucleotide (DFO) of the invention comprises a first region and a second region, wherein the second region comprises a nucleotide sequence comprising an inverted repeat of nucleotide sequence of the first region such that the DFO molecule can assemble into a double stranded oligonucleotide. Such double stranded oligonucleotides can act as a short interfering nucleic acid (siNA) to modulate gene expression. Each strand of the double stranded oligonucleotide duplex formed by DFO molecules of the invention can comprise a nucleotide sequence region that is complementary to the same nucleotide sequence in a target nucleic acid molecule (e.g., target interleukin and/or interleukin receptor RNA).

In one embodiment, the invention features a single stranded DFO that can assemble into a double stranded oligonucleotide. The applicant has surprisingly found that a single stranded oligonucleotide with nucleotide regions of self complementarity can readily assemble into duplex oligonucleotide constructs. Such DFOs can assemble into duplexes that can inhibit gene expression in a sequence specific manner. The DFO molecules of the invention comprise a first region with nucleotide sequence that is complementary to the nucleotide sequence of a second region and where the sequence of the first region is complementary to a target nucleic acid (e.g., RNA). The DFO can form a double stranded oligonucleotide wherein a portion of each strand of the double stranded oligonucleotide comprises a sequence complementary to a target nucleic acid sequence.

In one embodiment, the invention features a double stranded oligonucleotide, wherein the two strands of the double stranded oligonucleotide are not covalently linked to each other, and wherein each strand of the double stranded oligonucleotide comprises a nucleotide sequence that is complementary to the same nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., interleukin and/or interleukin receptor RNA target). In another embodiment, the two strands of the double stranded oligonucleotide share an identical nucleotide sequence of at least about 15, preferably at least about 16, 17, 18, 19, 20, or 21 nucleotides.

In one embodiment, a DFO molecule of the invention comprises a structure having Formula DFO-I:

wherein Z comprises a palindromic or repeat nucleic acid sequence optionally with one or more modified nucleotides (e.g., nucleotide with a modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length of about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 1 and about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein sequence X and Z, either independently or together, comprise nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence or a portion thereof (e.g., interleukin and/or interleukin receptor RNA target). For example, X independently can comprise a sequence from about 12 to about 21 or more (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) nucleotides in length that is complementary to nucleotide sequence in a target interleukin and/or interleukin receptor RNA or a portion thereof. In another non-limiting example, the length of the nucleotide sequence of X and Z together, when X is present, that is complementary to the target RNA or a portion thereof (e.g., interleukin and/or interleukin receptor RNA target) is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In yet another non-limiting example, when X is absent, the length of the nucleotide sequence of Z that is complementary to the target interleukin and/or interleukin receptor RNA or a portion thereof is from about 12 to about 24 or more nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24, or more). In one embodiment X, Z and X′ are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact (e.g., base pair) with a nucleotide sequence in the target RNA or a portion thereof (e.g., interleukin and/or interleukin receptor RNA target). In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In another embodiment, the lengths of oligonucleotides X and Z, or Z and X′, or X, Z and X′ are either identical or different.

When a sequence is described in this specification as being of “sufficient” length to interact (i.e., base pair) with another sequence, it is meant that the length is such that the number of bonds (e.g., hydrogen bonds) formed between the two sequences is enough to enable the two sequence to form a duplex under the conditions of interest. Such conditions can be in vitro (e.g., for diagnostic or assay purposes) or in vivo (e.g., for therapeutic purposes). It is a simple and routine matter to determine such lengths.

In one embodiment, the invention features a double stranded oligonucleotide construct having Formula DFO-I(a):

wherein Z comprises a palindromic or repeat nucleic acid sequence or palindromic or repeat-like nucleic acid sequence with one or more modified nucleotides (e.g., nucleotides with a modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein each X and Z independently comprises a nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof (e.g., interleukin and/or interleukin receptor RNA target) and is of length sufficient to interact with the target nucleic acid sequence of a portion thereof (e.g., interleukin and/or interleukin receptor RNA target). For example, sequence X independently can comprise a sequence from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) in length that is complementary to a nucleotide sequence in a target RNA or a portion thereof (e.g., interleukin and/or interleukin receptor RNA target). In another non-limiting example, the length of the nucleotide sequence of X and Z together (when X is present) that is complementary to the target interleukin and/or interleukin receptor RNA or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In yet another non-limiting example, when X is absent, the length of the nucleotide sequence of Z that is complementary to the target interleukin and/or interleukin receptor RNA or a portion thereof is from about 12 to about 24 or more nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24 or more). In one embodiment X, Z and X′ are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact (e.g., base pair) with nucleotide sequence in the target RNA or a portion thereof (e.g., interleukin and/or interleukin receptor RNA target). In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In another embodiment, the lengths of oligonucleotides X and Z or Z and X′ or X, Z and X′ are either identical or different. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a DFO molecule of the invention comprises structure having Formula DFO-II:

wherein each X and X′ are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises, for example, a nucleic acid sequence of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein X comprises a nucleotide sequence that is complementary to a target nucleic acid sequence (e.g., interleukin and/or interleukin receptor RNA) or a portion thereof and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence of a portion thereof. In one embodiment, the length of oligonucleotides X and X′ are identical. In another embodiment the length of oligonucleotides X and X′ are not identical. In one embodiment, length of the oligonucleotides X and X′ are sufficient to form a relatively stable double stranded oligonucleotide.

In one embodiment, the invention features a double stranded oligonucleotide construct having Formula DFO-II(a):

wherein each X and X′ are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein X comprises nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof (e.g., interleukin and/or interleukin receptor RNA target) and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence (e.g., interleukin and/or interleukin receptor RNA) or a portion thereof. In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In one embodiment, the lengths of the oligonucleotides X and X′ are sufficient to form a relatively stable double stranded oligonucleotide. In one embodiment, the double stranded oligonucleotide construct of Formula II(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, the invention features a DFO molecule having Formula DFO-I(b):

where Z comprises a palindromic or repeat nucleic acid sequence optionally including one or more non-standard or modified nucleotides (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) that can facilitate base-pairing with other nucleotides. Z can be, for example, of length sufficient to interact (e.g., base pair) with nucleotide sequence of a target nucleic acid (e.g., interleukin and/or interleukin receptor RNA) molecule, preferably of length of at least 12 nucleotides, specifically about 12 to about 24 nucleotides (e.g., about 12, 14, 16, 18, 20, 22 or 24 nucleotides). p represents a terminal phosphate group that can be present or absent.

In one embodiment, a DFO molecule having any of Formula DFO-I, DFO-I(a), DFO-I(b), DFO-II(a) or DFO-II can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.

In one embodiment, the palindrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of DFO constructs having Formula DFO-I, DFO-I(a) and DFO-I(b), comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non-Watson Crick base pairs).

In one embodiment, a DFO molecule of the invention, for example a DFO having Formula DFO-I or DFO-II, comprises about 15 to about 40 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, a DFO molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified 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 or in cells or tissues. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.

Multifunctional or Multi-Targeted siNA Molecules of the Invention

In one embodiment, the invention features siNA molecules comprising multifunctional short interfering nucleic acid (multifunctional siNA) molecules that modulate the expression of one or more genes in a biologic system, such as a cell, tissue, or organism. The multifunctional short interfering nucleic acid (multifunctional siNA) molecules of the invention can target more than one region a interleukin and/or interleukin receptor target nucleic acid sequence or can target sequences of more than one distinct target nucleic acid molecules, for example, interleukin and/or interleukin receptor, CHRM3 (see for example U.S. Ser. No. 10/919,866, incorporated by reference herein), ADAM33 (see for example U.S. Ser. No. 10/923,329, incorporated by reference herein), GPRA/AAA1 (see for example U.S. Ser. No. 10/923,182, incorporated by reference herein); and/or ADORA1 (see for example U.S. Ser. No. 10/224,005, incorporated by reference herein). The multifunctional siNA molecules of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The multifunctional siNA molecules of the instant invention provide useful reagents and methods for a variety of human applications, therapeutic, cosmetic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, referred to herein for convenience but not limitation as multifunctional short interfering nucleic acid or multifunctional siNA molecules, are potent mediators of sequence specific regulation of gene expression. The multifunctional siNA molecules of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides, etc.) in that they represent a class of polynucleotide molecules that are designed such that each strand in the multifunctional siNA construct comprises a nucleotide sequence that is complementary to a distinct nucleic acid sequence in one or more target nucleic acid molecules. A single multifunctional siNA molecule (generally a double-stranded molecule) of the invention can thus target more than one (e.g., 2, 3, 4, 5, or more) differing target nucleic acid target molecules. Nucleic acid molecules of the invention can also target more than one (e.g., 2, 3, 4, 5, or more) region of the same target nucleic acid sequence. As such multifunctional siNA molecules of the invention are useful in down regulating or inhibiting the expression of one or more target nucleic acid molecules. For example, a multifunctional siNA molecule of the invention can target nucleic acid molecules encoding interleukin and/or interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 targets. By reducing or inhibiting expression of more than one target nucleic acid molecule with one multifunctional siNA construct, multifunctional siNA molecules of the invention represent a class of potent therapeutic agents that can provide simultaneous inhibition of multiple targets within a disease or pathogen related pathway. Such simultaneous inhibition can provide synergistic therapeutic treatment strategies without the need for separate preclinical and clinical development efforts or complex regulatory approval process.

Use of multifunctional siNA molecules that target more then one region of a target nucleic acid molecule (e.g., messenger RNA) is expected to provide potent inhibition of gene expression. For example, a single multifunctional siNA construct of the invention can target both conserved and variable regions of a target nucleic acid molecule, such as interleukin and/or interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 target RNA or DNA, thereby allowing down regulation or inhibition of different splice variants encoded by a single gene, or allowing for targeting of both coding and non-coding regions of a target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotides where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are generally assembled from two separate oligonucleotides (e.g., siRNA). Alternately, a duplex can be formed from a single molecule that folds on itself (e.g., shRNA or short hairpin RNA). These double stranded oligonucleotides are known in the art to mediate RNA interference and all have a common feature wherein only one nucleotide sequence region (guide sequence or the antisense sequence) has complementarity to a target nucleic acid sequence, such as interleukin and/or interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 targets, and the other strand (sense sequence) comprises nucleotide sequence that is homologous to the target nucleic acid sequence. Generally, the antisense sequence is retained in the active RISC complex and guides the RISC to the target nucleotide sequence by means of complementary base-pairing of the antisense sequence with the target sequence for mediating sequence-specific RNA interference. It is known in the art that in some cell culture systems, certain types of unmodified siRNAs can exhibit “off target” effects. It is hypothesized that this off-target effect involves the participation of the sense sequence instead of the antisense sequence of the siRNA in the RISC complex (see for example Schwarz et al., 2003, Cell, 115, 199-208). In this instance the sense sequence is believed to direct the RISC complex to a sequence (off-target sequence) that is distinct from the intended target sequence, resulting in the inhibition of the off-target sequence. In these double stranded nucleic acid molecules, each strand is complementary to a distinct target nucleic acid sequence. However, the off-targets that are affected by these dsRNAs are not entirely predictable and are non-specific.

Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of down regulating or inhibiting the expression of more than one target nucleic acid sequence using a single multifunctional siNA construct. The multifunctional siNA molecules of the invention are designed to be double-stranded or partially double stranded, such that a portion of each strand or region of the multifunctional siNA is complementary to a target nucleic acid sequence of choice. As such, the multifunctional siNA molecules of the invention are not limited to targeting sequences that are complementary to each other, but rather to any two differing target nucleic acid sequences. Multifunctional siNA molecules of the invention are designed such that each strand or region of the multifunctional siNA molecule, that is complementary to a given target nucleic acid sequence, is of suitable length (e.g., from about 16 to about 28 nucleotides in length, preferably from about 18 to about 28 nucleotides in length) for mediating RNA interference against the target nucleic acid sequence. The complementarity between the target nucleic acid sequence and a strand or region of the multifunctional siNA must be sufficient (at least about 8 base pairs) for cleavage of the target nucleic acid sequence by RNA interference. multifunctional siNA of the invention is expected to minimize off-target effects seen with certain siRNA sequences, such as those described in (Schwarz et al., supra).

It has been reported that dsRNAs of length between 29 base pairs and 36 base pairs (Tuschl et al., International PCT Publication No. WO 02/44321) do not mediate RNAi. One reason these dsRNAs are inactive may be the lack of turnover or dissociation of the strand that interacts with the target RNA sequence, such that the RISC complex is not able to efficiently interact with multiple copies of the target RNA resulting in a significant decrease in the potency and efficiency of the RNAi process. Applicant has surprisingly found that the multifunctional siNAs of the invention can overcome this hurdle and are capable of enhancing the efficiency and potency of RNAi process. As such, in certain embodiments of the invention, multifunctional siNAs of length of about 29 to about 36 base pairs can be designed such that, a portion of each strand of the multifunctional siNA molecule comprises a nucleotide sequence region that is complementary to a target nucleic acid of length sufficient to mediate RNAi efficiently (e.g., about 15 to about 23 base pairs) and a nucleotide sequence region that is not complementary to the target nucleic acid. By having both complementary and non-complementary portions in each strand of the multifunctional siNA, the multifunctional siNA can mediate RNA interference against a target nucleic acid sequence without being prohibitive to turnover or dissociation (e.g., where the length of each strand is too long to mediate RNAi against the respective target nucleic acid sequence). Furthermore, design of multifunctional siNA molecules of the invention with internal overlapping regions allows the multifunctional siNA molecules to be of favorable (decreased) size for mediating RNA interference and of size that is well suited for use as a therapeutic agent (e.g., wherein each strand is independently from about 18 to about 28 nucleotides in length). Non-limiting examples are illustrated in FIGS. 16-28.

In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises a nucleotide sequence complementary to a nucleic acid sequence of a first target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleic acid sequence complementary to a nucleic acid sequence of a second target nucleic acid molecule. In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of the first region of a target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of a second region of a the target nucleic acid molecule. In another embodiment, the first region and second region of the multifunctional siNA can comprise separate nucleic acid sequences that share some degree of complementarity (e.g., from about 1 to about 10 complementary nucleotides). In certain embodiments, multifunctional siNA constructs comprising separate nucleic acid sequences can be readily linked post-synthetically by methods and reagents known in the art and such linked constructs are within the scope of the invention. Alternately, the first region and second region of the multifunctional siNA can comprise a single nucleic acid sequence having some degree of self complementarity, such as in a hairpin or stem-loop structure. Non-limiting examples of such double stranded and hairpin multifunctional short interfering nucleic acids are illustrated in FIGS. 16 and 17 respectively. These multifunctional short interfering nucleic acids (multifunctional siNAs) can optionally include certain overlapping nucleotide sequence where such overlapping nucleotide sequence is present in between the first region and the second region of the multifunctional siNA (see for example FIGS. 18 and 19).

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein each strand of the multifunctional siNA independently comprises a first region of nucleic acid sequence that is complementary to a distinct target nucleic acid sequence and the second region of nucleotide sequence that is not complementary to the target sequence. The target nucleic acid sequence of each strand is in the same target nucleic acid molecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence that is distinct from the target nucleotide sequence complementary to the first strand nucleotide sequence (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 1 of the first strand. The target nucleic acid sequence of complementary region 1 and complementary region 2 is in the same target nucleic acid molecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene, such as interleukin and/or interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1, (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene that is distinct from the gene of complementary region 1 (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 1 of the first strand.

In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a first gene, such as interleukin and/or interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1, (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a second target nucleic acid sequence distinct from the first target nucleic acid sequence of complementary region 1 (complementary region 2), provided, however, that the target nucleic acid sequence for complementary region 1 and target nucleic acid sequence for complementary region 2 are both derived from the same gene, and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to nucleotide sequence in the non-complementary region 1 of the first strand.

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having nucleotide sequence complementary to nucleotide sequence within a first target nucleic acid molecule, and in which the second sequence comprises a first region having nucleotide sequence complementary to a distinct nucleotide sequence within the same target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence.

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having a nucleotide sequence complementary to a nucleotide sequence within a first target nucleic acid molecule, and in which the second sequence comprises a first region having a nucleotide sequence complementary to a distinct nucleotide sequence within a second target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence.

In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises a nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a first target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within a second target nucleic acid molecule.

In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within the same target nucleic acid molecule.

In one embodiment, the invention features a double stranded multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein one strand of the multifunctional siNA comprises a first region having nucleotide sequence complementary to a first target nucleic acid sequence, and the second strand comprises a first region having a nucleotide sequence complementary to a second target nucleic acid sequence. The first and second target nucleic acid sequences can be present in separate target nucleic acid molecules or can be different regions within the same target nucleic acid molecule. As such, multifunctional siNA molecules of the invention can be used to target the expression of different genes, splice variants of the same gene, both mutant and conserved regions of one or more gene transcripts, or both coding and non-coding sequences of the same or differing genes or gene transcripts.

In one embodiment, a target nucleic acid molecule of the invention encodes a single protein. In another embodiment, a target nucleic acid molecule encodes more than one protein (e.g., 1, 2, 3, 4, 5 or more proteins). As such, a multifunctional siNA construct of the invention can be used to down regulate or inhibit the expression of several proteins. For example, a multifunctional siNA molecule comprising a region in one strand having nucleotide sequence complementarity to a first target nucleic acid sequence derived from a gene encoding one protein and the second strand comprising a region with nucleotide sequence complementarity to a second target nucleic acid sequence present in target nucleic acid molecules derived from genes encoding two or more proteins (e.g., two or more differing interleukin, interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 target sequences) can be used to down regulate, inhibit, or shut down a particular biologic pathway by targeting, for example, two or more targets involved in a biologic pathway.

In one embodiment the invention takes advantage of conserved nucleotide sequences present in different isoforms of cytokines or ligands and receptors for the cytokines or ligands. By designing multifunctional siNAs in a manner where one strand includes a sequence that is complementary to a target nucleic acid sequence conserved among various isoforms of a cytokine and the other strand includes sequence that is complementary to a target nucleic acid sequence conserved among the receptors for the cytokine, it is possible to selectively and effectively modulate or inhibit a biological pathway or multiple genes in a biological pathway using a single multifunctional siNA.

In one embodiment, a double stranded multifunctional siNA molecule of the invention comprises a structure having Formula MF-I:

wherein each 5′-p-XZX′-3′ and 5′-p-YZY′-3′ are independently an oligonucleotide of length of about 20 nucleotides to about 300 nucleotides, preferably of about 20 to about 200 nucleotides, about 20 to about 100 nucleotides, about 20 to about 40 nucleotides, about 20 to about 40 nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38 nucleotides; XZ comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence; YZ is an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence; Z comprises nucleotide sequence of length about 1 to about 24 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides) that is self complimentary; X comprises nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that is complementary to nucleotide sequence present in region Y′; Y comprises nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1- about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) that is complementary to nucleotide sequence present in region X′; each p comprises a terminal phosphate group that is independently present or absent; each XZ and YZ is independently of length sufficient to stably interact (i.e., base pair) with the first and second target nucleic acid sequence, respectively, or a portion thereof. For example, each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as target RNAs or a portion thereof. In another non-limiting example, the length of the nucleotide sequence of X and Z together that is complementary to the first target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In another non-limiting example, the length of the nucleotide sequence of Y and Z together, that is complementary to the second target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., interleukin and/or interleukin receptor RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., interleukin, interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 targets). In one embodiment, Z comprises a palindrome or a repeat sequence. In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In one embodiment, the lengths of oligonucleotides Y and Y′ are identical. In another embodiment, the lengths of oligonucleotides Y and Y′ are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-II:

wherein each 5′-p-XX′-3′ and 5′-p-YY′-3′ are independently an oligonucleotide of length of about 20 nucleotides to about 300 nucleotides, preferably about 20 to about 200 nucleotides, about 20 to about 100 nucleotides, about 20 to about 40 nucleotides, about 20 to about 40 nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38 nucleotides; X comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence; Y is an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence; X comprises a nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that is complementary to nucleotide sequence present in region Y′; Y comprises nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) that is complementary to nucleotide sequence present in region X′; each p comprises a terminal phosphate group that is independently present or absent; each X and Y independently is of length sufficient to stably interact (i.e., base pair) with the first and second target nucleic acid sequence, respectively, or a portion thereof. For example, each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as interleukin, interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 target RNAs or a portion thereof. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., interleukin and/or interleukin receptor RNA or DNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules, such as interleukin, interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 target sequences or a portion thereof. In one embodiment, Z comprises a palindrome or a repeat sequence. In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In one embodiment, the lengths of oligonucleotides Y and Y′ are identical. In another embodiment, the lengths of oligonucleotides Y and Y′ are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-III:

wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y′; X′ comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each X and X′ is independently of length sufficient to stably interact (i.e., base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y; and the multifunctional siNA directs cleavage of the first and second target sequence via RNA interference. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., interleukin and/or interleukin receptor RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules such as interleukin, interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 target sequences or a portion thereof. In one embodiment, region W connects the 3′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, region W connects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X′. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y′. In one embodiment, W connects sequences Y and Y′ via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-IV:

wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y′; X′ comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each Y and Y′ is independently of length sufficient to stably interact (i.e., base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y; and the multifunctional siNA directs cleavage of the first and second target sequence via RNA interference. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., interleukin and/or interleukin receptor RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules, such as interleukin, interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 target sequences or a portion thereof. In one embodiment, region W connects the 3′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, region W connects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X′. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y′. In one embodiment, W connects sequences Y and Y′ via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-V:

wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y′; X′ comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each X, X′, Y, or Y′ is independently of length sufficient to stably interact (i.e., base pair) with a first, second, third, or fourth target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y; and the multifunctional siNA directs cleavage of the first, second, third, and/or fourth target sequence via RNA interference. In one embodiment, the first, second, third and fourth target nucleic acid sequence are all present in the same target nucleic acid molecule (e.g., interleukin and/or interleukin receptor RNA). In another embodiment, the first, second, third and fourth target nucleic acid sequence are independently present in different target nucleic acid molecules, such as interleukin, interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 target sequences or a portion thereof. In one embodiment, region W connects the 3′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, region W connects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X′. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y′. In one embodiment, W connects sequences Y and Y′ via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, regions X and Y of multifunctional siNA molecule of the invention (e.g., having any of Formula MF-1-MF-V), are complementary to different target nucleic acid sequences that are portions of the same target nucleic acid molecule. In one embodiment, such target nucleic acid sequences are at different locations within the coding region of a RNA transcript. In one embodiment, such target nucleic acid sequences comprise coding and non-coding regions of the same RNA transcript. In one embodiment, such target nucleic acid sequences comprise regions of alternately spliced transcripts or precursors of such alternately spliced transcripts.

In one embodiment, a multifunctional siNA molecule having any of Formula MF-1-MF-V can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII described herein, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.

In one embodiment, the palindrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of multifunctional siNA constructs having Formula MF-I or MF-II comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non-Watson Crick base pairs).

In one embodiment, a multifunctional siNA molecule of the invention, for example each strand of a multifunctional siNA having MF-I-MF-V, independently comprises about 15 to about 40 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, a multifunctional siNA molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified 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 or in cells or tissues. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.

In another embodiment, the invention features multifunctional siNAs, wherein the multifunctional siNAs are assembled from two separate double-stranded siNAs, with one of the ends of each sense strand is tethered to the end of the sense strand of the other siNA molecule, such that the two antisense siNA strands are annealed to their corresponding sense strand that are tethered to each other at one end (see FIG. 22). The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one antisense strand of the siNA is tethered to the 3′-end of the antisense strand of the other siNA molecule, such that the 5′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand (see FIG. 22 (G-H)). In one embodiment, the linkage between the 5′-end of the first antisense strand and the 3′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′ end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interference-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one antisense strand of the siNA is tethered to the 5′-end of the antisense strand of the other siNA molecule, such that the 3′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand (see FIG. 22 (E)). In one embodiment, the linkage between the 5′-end of the first antisense strand and the 5′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′ end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interference-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 3′-end of one antisense strand of the siNA is tethered to the 3′-end of the antisense strand of the other siNA molecule, such that the 5′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand (see FIG. 22 (F)). In one embodiment, the linkage between the 5′-end of the first antisense strand and the 5′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′ end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interference-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In any of the above embodiments, a first target nucleic acid sequence or second target nucleic acid sequence can independently comprise interleukin, interleukin receptor, CHRM3, ADAM33, GPRA/AAA1, and/or ADORA1 RNA, DNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a interleukin and/or interleukin receptor RNA, DNA or a portion thereof and the second target nucleic acid sequence is a interleukin and/or interleukin receptor RNA, DNA of a portion thereof. In one embodiment, the first target nucleic acid sequence is a first interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-S, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, or IL-27) or interleukin receptor (e.g., IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26R, or IL-27R) RNA, DNA or a portion thereof and the second target nucleic acid sequence is a second interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, or IL-27) or interleukin receptor (e.g., IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26R, or IL-27R) RNA, DNA of a portion thereof. In one embodiment, the first target nucleic acid sequence is a interleukin and/or interleukin receptor RNA, DNA or a portion thereof and the second target nucleic acid sequence is a CHRM3 RNA, DNA of a portion thereof. In one embodiment, the first target nucleic acid sequence is a interleukin and/or interleukin receptor RNA, DNA or a portion thereof and the second target nucleic acid sequence is a GPRA/AAA1 RNA, DNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a interleukin and/or interleukin receptor RNA, DNA or a portion thereof and the second target nucleic acid sequence is an ADORA1 RNA, DNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a interleukin and/or interleukin receptor RNA, DNA or a portion thereof and the second target nucleic acid sequence is an ADAM33 RNA, DNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a IL-4 RNA, DNA or a portion thereof and the second target nucleic acid sequence is an IL-4R RNA, DNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a IL-13 RNA, DNA or a portion thereof and the second target nucleic acid sequence is an IL-13R RNA, DNA or a portion thereof.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in 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 min 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 Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.

The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min 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 Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. 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 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO:1/1 (0.8 mL) at 65° C. for 15 minutes. The vial is brought to room temperature TEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 minutes. The sample is cooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al, 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.

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.

The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, 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.

In another aspect of the invention, siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 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.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in viva 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.

In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see for example Wengel et al, International PCT Publication No. WO 00/66604 and WO 99/14226).

In another embodiment, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation.

The term “biologically active molecule” as used herein refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead to better treatments by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more 5′ and/or a 3′-cap structure, for example, on only the sense siNA strand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or may be present on both termini. In non-limiting examples, the 5′-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety. Non-limiting examples of cap moieties are shown in FIG. 10.

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).

By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, —O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino or SH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers, to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR, where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.

In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, 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.

By “abasic” is meant sugar moieties lacking a nucleobase or having a hydrogen atom (H) or other non-nucleobase chemical groups in place of a nucleobase at the 1′ position of the sugar moiety, see for example Adamic et al., U.S. Pat. No. 5,998,203. In one embodiment, an abasic moiety of the invention is a ribose, deoxyribose, or dideoxyribose sugar.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1′ carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′—NH₂or 2′-O—NH₂, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.

Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to prevent or treat cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, and/or proliferative diseases, conditions, disorders, traits and/or conditions described herein or otherwise known in the art to be related to gene expression, and/or any other trait, disease, disorder or condition that is related to or will respond to the levels of interleukin and/or interleukin receptor in a cell or tissue, alone or in combination with other therapies.

In one embodiment a siNA composition of the invention 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; Wang et al, International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example 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). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acid molecules of the invention are formulated as described in United States Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety.

In one embodiment, a siNA molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.), all of which are incorporated by reference herein in their entirety. Such siNA formulations are generally referred to as “lipid nucleic acid particles” (LNP).

In one embodiment, a siNA molecule of the invention is complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666, incorporated by reference herein in its entirety including the drawings. In another embodiment, the membrane disruptive agent or agents and the siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Pat. No. 6,235,310, incorporated by reference herein in its entirety including the drawings.

In one embodiment, a siNA molecule of the invention is complexed with delivery systems as described in U.S. Patent Application Publication No. 2003077829 and International PCT Publication Nos. WO 00/03683 and WO 02/087541, all incorporated by reference herein in their entirety including the drawings.

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

Aerosols of liquid particles comprising a nucleic acid composition of the invention can be produced by any suitable means, such as with a nebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers are commercially available devices which transform solutions or suspensions of an active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers comprise the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride or other suitable salts. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants. The aerosols of solid particles comprising the active composition and surfactant can likewise be produced with any solid particulate aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a therapeutic composition at a rate suitable for human administration.

In one embodiment, a solid particulate aerosol generator of the invention is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which can be delivered by means of an insufflator. In the insufflator, the powder, e.g., a metered dose thereof effective to carry out the treatments described herein, is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation can additionally contain one or more co-solvents, for example, ethanol, emulsifiers and other formulation surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents. Other methods for pulmonary delivery are described in, for example US Patent Application No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728; 6,565,885, all incorporated by reference herein.

In one embodiment, the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to the central nervous system and/or peripheral nervous system. Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a 15 mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express repeat expansion allelic variants for modulation of RE gene expression. The delivery of nucleic acid molecules of the invention, targeting RE is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.

In one embodiment, nucleic acid molecules of the invention are administered to the central nervous system (CNS) or peripheral nervous system (PNS). Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a 15 mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al, 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells in the CNS and/or PNS.

The delivery of nucleic acid molecules of the invention to the CNS is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.

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

In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered directly or topically (e.g., locally) to the dermis or follicles as is generally known in the art (see for example Brand, 2001, Curr. Opin. Mol. Ther., 3, 244-8; Regnier et al., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs, 16, 339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; and Preat and Dujardin, 2001, STP PharmaSciences, 11, 57-68; and Vogt et al., 2003, Hautarzt. 54, 692-8). In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered directly or topically using a hydroalcoholic gel formulation comprising an alcohol (e.g., ethanol or isopropanol), water, and optionally including additional agents such isopropyl myristate and carbomer 980.

In one embodiment, delivery systems of the invention include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).

In one embodiment, delivery systems of the invention include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, a siNA molecule of the invention is administered iontophoretically, for example to the dermis or to other relevant tissues. Non-limiting examples of iontophoretic delivery are described in, for example, WO 03/043689 and WO 03/030989, which are incorporated by reference in their entireties herein.

In one embodiment, siNA molecules of the invention are formulated or complexed with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine derivatives, including for example grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPA Pharm Sci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kuiath et al., 2002, Pharmaceutical Research, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release, 60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; and Sagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises a bioconjugate, for example a nucleic acid conjugate as described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S. Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,138,045, all incorporated by reference herein.

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 to 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 creams, gels, sprays, oils and other suitable compositions for topical, dermal, or transdermal administration as is known in the art.

The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, 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.

In one embodiment, siNA molecules of the invention are administered to a subject by systemic administration in a pharmaceutically acceptable composition or formulation. 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, portal vein, intraperitoneal, inhalation, nebulization, 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.

By “pharmaceutically acceptable formulation” or “pharmaceutically acceptable composition” 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); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of a composition comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) and nucleic acid molecules of the invention. These formulations offer a method for increasing the accumulation of drugs (e.g., siNA) 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, Biochem. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, 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.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

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

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

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

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

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

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

Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). In another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser. No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., 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 a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al, 1996, TIG., 12, 510).

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

In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siNA of the invention; and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev, 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al, 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736. The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.

In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.

In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.

Interleukin and Interleukin Receptor Biology and Biochemistry

The following discussion is adapted from R&D Systems Mini-Reveiws and Tech Notes, Cytokine Mini-Reviews, Copyright ©2002 R&D Systems. Interleukin 2 (IL-2) is a lymphokine synthesized and secreted primarily by T helper lymphocytes that have been activated by stimulation with certain mitogens or by interaction of the T cell receptor complex with antigen/MHC complexes on the surfaces of antigen-presenting cells. The response of T helper cells to activation is induction of the expression of IL-2 and receptors for IL-2 and, subsequently, clonal expansion of antigen-specific T cells. At this level IL-2 is an autocrine factor, driving the expansion of the antigen-specific cells. IL-2 also acts as a paracrine factor, influencing the activity of other cells, both within the immune system and outside of it. B cells and natural killer (NK) cells respond, when properly activated, to IL-2. The so-called lymphocyte activated killer, or LAK cells, appear to be derived from NK cells under the influence of IL-2.

The biological activities of IL-2 are mediated through the binding of IL-2 to a multisubunit cellular receptor. Although three distinct transmembrane glycoprotein subunits contribute to the formation of the high affinity IL-2 receptor, various combinations of receptor subunits (alpha, beta, gamma) are known to occur.

Interleukin 1 (IL-1) is a general name for two distinct proteins, IL-1a and IL-1b, that are considered the first of a family of regulatory and inflammatory cytokines. Along with IL-1 receptor antagonist (IL-1ra)₂and IL-18,3 these molecules play important roles in the up- and down-regulation of acute inflammation. In the immune system, the production of IL-1 is typically induced, generally resulting in inflammation. IL-1b and TNF-a are generally thought of as prototypical pro-inflammatory cytokines. The effects of IL-1, however, are not limited to inflammation, as IL-1 has also been associated with bone formation and remodeling, insulin secretion, appetite regulation, fever induction, neuronal phenotype development, and IGF/GH physiology. IL-1 has also been known by a number of alternative names, including lymphocyte activating factor, endogenous pyrogen, catabolin, hemopoietin-1, melanoma growth inhibition factor, and osteoclast activating factor. IL-1a and IL-1b exert their effects by binding to specific receptors. Two distinct IL-1 receptor binding proteins, plus a non-binding signaling accessory protein have been identified to date. Each have three extracellular immunoglobulin-like (Ig-like) domains, qualifying them for membership in the type IV cytokine receptor family.

Interleukin-4 (IL-4) mediates important pro-inflammatory functions in asthma including induction of the IgE isotype switch, expression of vascular cell adhesion molecule-1 (VCAM-1), promotion of eosinophil transmigration across endothelium, mucus secretion, and differentiation of T helper type 2 lymphocytes leading to cytokine release. Asthma has been linked to polymorphisms in the IL-4 gene promoter and proteins involved in IL-4 signaling. Soluble recombinant IL-4 receptor lacks transmembrane and cytoplasmic activating domains and can therefore sequester IL-4 without mediating cellular activation. Genetic variants within the IL-4 signalling pathway might contribute to the risk of developing asthma in a given individual. A number of polymorphisms have been described within the IL-4 receptor a (IL-4Rα) gene, and in addition, polymorphism occurs in the promoter for the IL-4 gene itself (see for example Hall, 2000, Respir. Res., 1, 6-8 and Ober et al., 2000, Am J Hum Genet., 66, 517-526, for a review). The type 2 cytokine IL-13, which shares a receptor component and signaling pathways with IL-4, was found to be necessary and sufficient for the expression of allergic asthma (see Wills-Karp et al., 1998, Science, 282, 2258-61). IL-13 induces the pathophysiological features of asthma in a manner that is independent of immunoglobulin E and eosinophils. Thus, IL-13 is critical to allergen-induced asthma but operates through mechanisms other than those that are classically implicated in allergic responses.

Human IL-5 is a 134 amino acid polypeptide with a predicted mass of 12.5 kDa. It is secreted by a restricted number of mesenchymal cell types. In its native state, mature IL-5 is synthesized as a 115 aa, highly glycosylated 22 kDa monomer that forms a 40-50 kDa disulfide-linked homodimer. Although the content of carbohydrate is high, carbohydrate is not needed for bioactivity. Monomeric IL-5 has no activity; a homodimer is required for function. This is in contrast to the receptor-related cytokines IL-3 and GM-CSF, which exist only as monomers. Just as one IL-3 and GM-CSF monomer binds to one receptor, one IL-5 homodimer is able to engage only one IL-5 receptor. It has been suggested that IL-5 (as a dimer) undergoes a general conformational change after binding to one receptor molecule, and this change precludes binding to a second receptor. The receptor for IL-5 consists of a ligand binding a-subunit and a non-ligand binding (common) signal transducing b-subunit that is shared by the receptors for IL-3 and GM-CSF. IL-5 appears to perform a number of functions on eosinophils. These include the down modulation of Mac-1, the upregulation of receptors for IgA and IgG, the stimulation of lipid mediator (leukotriene C4 and PAF) secretion and the induction of granule release. IL-5 also promotes the growth and differentiation of eosinophils.

Interleukin 6 (IL-6) is considered a prototypic pleiotrophic cytokine. This is reflected in the variety of names originally assigned to IL-6 based on function, including Interferon b2, IL-1-inducible 26 kD Protein, Hepatocyte Stimulating Factor, Cytotoxic T-cell Differentiation Factor, B cell Differentiation Factor (BCDF) and/or B cell Stimulatory Factor 2 (BSF2). A number of cytokines make up an IL-6 cytokine family. Membership in this family is typically based on a helical cytokine structure and receptor subunit makeup. The functional receptor for IL-6 is a complex of two transmembrane glycoproteins (gp130 and IL-6 receptor) that are members of the Class I cytokine receptor superfamily.

Because of the central role of the interleukin family of cytokines in the mediation of immune and inflammatory responses, modulation of interleukin expression and/or activity can provide important functions in therapeutic and diagnostic applications. The use of small interfering nucleic acid molecules targeting interleukins and their corresponding receptors therefore provides a class of novel therapeutic agents that can be used in the treatment of cancers, proliferative diseases, inflammatory disease, respiratory disease, pulmonary disease, cardiovascular disease, autoimmune disease, neurologic disease, infectious disease, prior disease, renal disease, transplant rejection, or any other disease or condition that responds to modulation of interleukin and interleukin receptor genes.

EXAMPLES

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

Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complement in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises 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.

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.

Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example, using a Waters C18 SepPak 1 g 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.

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

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, trait, 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

The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.
2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.
3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As 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.
4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.
5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.
6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.
7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide TU (uridine dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end of the sequence (to yield 3′ UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.
8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Tables II and II). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.
9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.
10. Other design considerations can be used when selecting target nucleic acid sequences, see, for example, Reynolds et al., 2004, Nature Biotechnology Advanced Online Publication, 1 Feb. 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a interleukin and/or interleukin receptor target sequence is used to screen for target sites in cells expressing interleukin and/or interleukin receptor RNA, such as cultured Jurkat, HeLa, A549 or 293T cells. The general strategy used in this approach is shown in FIG. 9. A non-limiting example of such is a pool comprising sequences having any of SEQ ID NOS 1-1260 and 1269-2358. Cells expressing interleukin and/or interleukin receptor are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor mRNA levels or decreased interleukin and/or interleukin receptor protein expression), are sequenced to determine the most suitable target site(s) within the target interleukin and/or interleukin receptor RNA sequence.

Example 4
Interleukin and/or Interleukin Receptor Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the interleukin and/or interleukin receptor RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein. siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constructs that possess both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for example FIG. 11).

Example 5
Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence. Generally, siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein in their entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl, 3′-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2′-O—Silyl Ethers can be used in conjunction with acid-labile 2′-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2′ chemistries can require different protecting groups, for example 2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′- to 5′-direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support (e.g., controlled pore glass or polystyrene) using various linkers. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5′-end of the first nucleoside. The support is then washed and any unreacted 5′-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage. At the end of the nucleotide addition cycle, the 5′-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.

Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in 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

An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor 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.

Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-³²P] CTP, passed over a G50 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 IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites in the interleukin and/or interleukin receptor RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the interleukin and/or interleukin receptor 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 Interleukin and/or Interleukin Receptor Target RNA In Vivo

siNA molecules targeted to the human interleukin and/or interleukin receptor 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 interleukin and/or interleukin receptor RNA are given in Table II and III.

Two formats are used to test the efficacy of siNAs targeting interleukin and/or interleukin receptor. First, the reagents are tested in cell culture using, for example, Jurkat, HeLa, A549 or 293T cells, to determine the extent of RNA and protein inhibition. siNA reagents (e.g.; see Tables II and III) are selected against the interleukin and/or interleukin receptor target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, Jurkat, HeLa, A549 or 293T cells. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 TAQMAN®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead siNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., Jurkat, HeLa, A549 or 293T cells) are seeded, for example, at 1×10⁵cells per well of a six-well dish in EGM-2 (BioWhittaler) 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 minutes in polystyrene tubes. Following vortexing, the complexed siNA is added to each well and incubated for the times indicated. For initial optimization experiments, cells are seeded, for example, at 1×10³in 96 well plates and siNA complex added as described. Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid. Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and Lightcycler Quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example, using Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays. For TAQMAN® analysis (real-time PCR monitoring of amplification), 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 μM each DATP, dCTP, dGTP, and dTTP, 10 U RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD® (DNA polymerase) (PE-Applied Biosystems) and 10 U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 minutes at 48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 600C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/reaction) and normalizing to β-actin or GAPDH mRNA in parallel TAQMAN® reactions (real-time PCR monitoring of amplification). 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 cRNA. Values are represented as relative expression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4° C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).

Example 8
Animal Models Useful to Evaluate the Down-Regulation of Interleukin and/or Interleukin Receptor Gene Expression

Evaluating the efficacy of anti-interleukin agents in animal models is an important prerequisite to human clinical trials. Allogeneic rejection is the most common cause of corneal graft failure. King et al., 2000, Transplantation, 70, 1225-1233, describe a study investigating the kinetics of cytokine and chemokine mRNA expression before and after the onset of corneal graft rejection. Intracorneal cytokine and chemokine mRNA levels were investigated in the Brown Norway-Lewis inbred rat model, in which rejection onset is observed at 8/9 days after grafting in all animals. Nongrafted corneas and syngeneic (Lewis-Lewis) corneal transplants were used as controls. Donor and recipient cornea were examined by quantitive competitive reverse transcription-polymerase chain reaction (RT-PCR) for hypoxyanthine phosphoribosyltransferase (HPRT), CD3, CD25, interleukin (IL)-1beta, IL-IRA, IL-2, IL-6, IL-10, interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), transforming growth factor (TGF)-beta1, and macrophage inflammatory protein (MIP)-2 and by RT-PCR for IL-4, IL-5, IL-12 p40, IL-13, TGF-beta.2, monocyte chemotactic protein-1 (MCP-1), MIP-1alpha, MIP-1beta, and RANTES. A biphasic expression of cytokine and chemokine mRNA was found after transplantation. During the early phase (days 3-9), there was an elevation of the majority of the cytokines examined, including IL-1beta, IL-6, IL-10, IL-12 p40, and MIP-2. There was no difference in cytokine expression patterns between allogeneic or syngeneic recipients at this time. In syngeneic recipients, cytokine levels reduced to pretransplant levels by day 13, whereas levels of all cytokines rose after the rejection onset in the allografts, including TGF-beta.1, TGF-beta.2, and IL-IRA. The T cell-derived cytokines IL-4, IL-13, and IFN-gamma were detected only during the rejection phase in allogeneic recipients. Thus, there appears to be an early cytokine and chemokine response to the transplantation process, evident in syngeneic and allogeneic grafts, that drives angiogenesis, leukocyte recruitment, and affects other leukocyte functions. After an immune response has been generated, allogeneic rejection results in the expression of Th1 cytokines, Th2 cytokines, and anti-inflammatory/Th3 cytokines. This animal model can be used to evaluate the efficacy of nucleic acid molecules of the invention targeting interleukin expression (e.g., phenotypic change, interleuking expression etc.) toward therapeutic use in treating transplant rejection. Similarly, other animal models of transplant rejection as are known in the art can be used to evaluate nucleic acid molecules (e.g., siNA) of the invention toward therapeutic use.

Other animal models are useful in evaluating the role of interleukins in asthma. For example, Kuperman et al., 2002, Nature Medicine, 8, 885-9, describe an animal model of IL-13 mediated asthma response animal models of allergic asthma in which blockade of IL-13 markedly inhibits allergen-induced asthma. Venkayya et al., 2002, Am J Respir Cell Mol. Biol., 26, 202-8 and Yang et al., 2001, Am J Respir Cell Mol. Biol., 25, 522-30 describe animal models of airway inflammation and airway hyperresponsiveness (AHR) in which IL-4/IL-4R and IL-13 mediate asthma. These models can be used to evaluate the efficacy of siNA molecules of the invention targeting, for example, IL-4, IL-4R, IL-13, and/or IL-13R for use is treating asthma.

Identification of Active siNA's in Cell Culture and Subsequent Evaluation of Synthetic siNA in Lung for Application to Respiratory Diseases Such as Asthma: Pulmonary-Distribution and Efficacy

The allergic inflammatory response leading to airway hyperesponssiveness is orchestrated by multiple mediators, including interleukins. An animal model of airway hyperresponsiveness following allergen challenge is used to evaluate the efficacy of siNA molecules of the invention designed to down regulate expression of interleukin and interleukin receptor targets, including IL-4, IL-4R, IL-13, and IL-13R. Several endpoints are evaluated following siNA treatment of allergen challenged animals compared to relevant controls, including lung function, IFN-alpha, IL-1, IL-5, IL-13, IL-10 and IL-12 protein levels in bronchial/alveolar lavage fluid as determined by ELISA. Counts of inflammatory cells including lymphocytes, neutrophils, macrophages, and eosinophils in bronchial/alveolar lavage fluid are taken. Histology is performed to evaluate end-points related to lung function including include thickening of the endothelial cell wall, mucous secretion, goblet cell hyperplasia, and the presence of eosinophils. Levels of IL-4, IL-5, and IL-13 mRNA in lung tissue are evaluated via quantitative PCR (TaqMan).

Active siNA constructs were identified in cell culture experiments using a dual luciferase reporter system (Promega, Madison, Wis.). The rat IL-4 and IL-13 genes were cloned into the 3′ untranslated region of Renilla luciferase to create a reporter plasmid. Specific siNA-induced degradation of the target sequence in Renilla mRNA transcribed from this plasmid results in a loss of Renilla luciferase signal in plasmid-transfected HeLa cells. The reporter plasmid also contains a copy of the Firefly luciferase gene, which does not contain the target site sequences. In HeLa cells co-transfected with the reporter plasmid and siNAs, the ratio of Renilla to Firefly luciferase activities (using two different substrates) provides a measure of siNA activity. The Firefly luciferase activity provides an internal control for transfection efficiency, toxicity and sample recovery. Using this reporter system, the inhibition of Renilla luciferase by siNAs targeting IL-4 (FIG. 29) and IL-13 (FIG. 30) was examined at a dose of 12.5 nM. As shown in FIGS. 29 and 30, Renilla luciferase activity was dramatically reduced by treatment with several siNA constructs (all greater than 70%). There was little to no inhibitory effect when the inverted control or an irrelevant siNA were tested at 12.5 mM. The most active sequences have IC₅₀s of 300 picomolar in this assay.

Following identification of active siNA constructs in vitro, a murine model of airway hyperresponsiveness (AHR) was used to assess the effectiveness of siNA's targeting IL-4, IL-4R, IL-13, and IL-13R in mitigating the inflammatory response after an allergic challenge. Assessment of multiple cytokine target mRNA and protein levels, as well as lung function endpoints allow a robust assessment siNA silencing activity in this model. Although IV injection was used for the delivery of siNA in the current study, the model is also ammenable to the use of siNA that is nebulized or delivered in a aerosolized formulation. The ability to deliver via several modalities makes possible the subsequent evaluation of efficacy following delivery by these methods

In a non-limiting example, 8 to 12 week old BalbC mice were be sensitized by i.p. injection with 20 μg OVA emulsified in 2.25 mg aluminum hydroxide in a total volume of 100 μl on days 1 and 14. Mice were challenged on three consecutive days (days 28, 29, 30) (20 min) via the airways with OVA (1% in normal saline) using ultrasonic nebulization (primary challenge). In the secondary challenge protocol, six weeks after the primary challenge, mice were exposed to a single OVA challenge (1% in normal saline). Administration of siNAs (Table III) was performed by injection into the tail vein. In the current study, a secondary challenge protocol was used and siNAs were administered 72, 48, and 3 hours prior to secondary challenge. In each dose, mice were administered either 30 μg of anti-IL-13 siNA mixed with 30 μg of anti-IL-4R siNA, 30 μg of anti-IL-13R siNA mixed with 30 μg of anti-IL-4R siNA, or 30 μg of each of two irrelevant siNAs. Twelve mice were tested for each group. Administration times of the siNAs can be varied.

Forty-eight hours following the last challenge airway responsiveness was assessed. Mice were anesthetized with pentobarbital sodium (70-90 mg/kg), tracheostomized and mechanically ventilated. Airway function was measured after challenge with aerosolized methacholine (MCh) via the airways for 10 sec (60 breaths/min, 500-μl tidal volume) in increasing concentrations (1.56, 3.13, 6.25, and 12.5 mg/ml). Immediately after assessment of lung function, lungs were lavaged via the tracheal tube with PBS (1 ml) and differential cell counts were performed. Mice receiving active siNA 38016/38138 and 37910/37958 targeting IL-13 and IL-4R or 37910/37958 and 38195/38243 targeting IL-4R and IL-13R formulated with polyethyleneimine (PEI) showed improved lung function compared to a matched chemistry siNA irrelevant sequence control.

One-half of the lungs were harvested for mRNA isolation. RT-PCR is used to determine mRNA levels of IL-4, IL-4R, IL-13, IL-13R and IFN-alpha. In addition, IFN-alpha, IL-4, IL-5, IL-13, IL-10, IL-12 levels in the BAL fluid are measured by ELISA. The other half of the harvested lungs were inflated and fixed with 10% formalin for histology.

Example 9
RNAi Mediated Inhibition of Interleukin and Interleukin Receptor Expression in Cell Culture Experiments

siNA constructs (Table III) are tested for efficacy in reducing interleukin and/or interleukin receptor RNA expression in, for example, Jurkat, HeLa, A549, or 293T 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 is determined.

In a non-limiting example, chemically modified siNA constructs (Table III) were tested for efficacy as described above in reducing IL-4R RNA expression in HeLa cells. Active siNAs were evaluated compared to untreated cells and a matched chemistry irrelevant control. Results are summarized in FIG. 29. FIG. 29 shows results for chemically modified siNA constructs targeting various sites in IL-4R RNA. As shown in FIG. 29, the active siNA constructs provide significant inhibition of IL-4R gene expression in cell culture experiments as determined by levels of IL-4R mRNA when compared to appropriate controls.

In another non-limiting example, chemically modified siNA constructs (Table III) were tested for efficacy as described above in reducing IL-13R RNA expression in HeLa cells. Active siNAs were evaluated compared to untreated cells and a matched chemistry irrelevant control. Results are summarized in FIG. 30. FIG. 30 shows results for chemically modified siNA constructs targeting various sites in IL-13R RNA. As shown in FIG. 30, the active siNA constructs provide significant inhibition of IL-13R gene expression in cell culture experiments as determined by levels of IL-13R mRNA when compared to appropriate controls.

Example 10
Indications

The siNA molecule of the invention can be used to prevent, inhibit or treat cancers and other proliferative conditions, viral infection, inflammatory disease, autoimmunity, respiratory disease, pulmonary disease, cardiovascular disease, neurologic disease, renal disease, ocular disease, liver disease, mitochondrial disease, endocrine disease, prion disease, reproduction related diseases and conditions, and/or any other trait, disease or condition that is related to or will respond to the levels of interleukin and/or interleukin receptor in a cell or tissue, alone or in combination with other treatments or therapies. Non-limiting examples of respiratory diseases that can be treated using siNA molecules of the invention (e.g., siNA molecules targeting IL-4, IL-4R, IL-13, and/or IL-13R include asthma, chronic obstructive pulmonary disease or “COPD”, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergies, impeded respiration, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema.

The use of anticholinergic agents, anti-inflammatories, bronchodilators, adenosine inhibitors, adenosine A1 receptor inhibitors, non-selective M3 receptor antagonists such as atropine, ipratropium brominde and selective M3 receptor antagonists such as darifenacin and revatropate are all non-limiting examples of agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention. Immunomodulators, chemotherapeutics, anti-inflammatory compounds, and anti-viral compounds are additional 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 prevention or treatment of traits, diseases and disorders herein. Those skilled in the art will recognize that other drug compounds and therapies can 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.

Example 11
Multifunctional siNA Inhibition of Interleukin and/or Interleukin Receptor RNA Expression

Multifunctional siNA Design

Once target sites have been identified for multifunctional siNA constructs, each strand of the siNA is designed with a complementary region of length, for example, of about 18 to about 28 nucleotides, that is complementary to a different target nucleic acid sequence. Each complementary region is designed with an adjacent flanking region of about 4 to about 22 nucleotides that is not complementary to the target sequence, but which comprises complementarity to the complementary region of the other sequence (see for example FIG. 16). Hairpin constructs can likewise be designed (see for example FIG. 17). Identification of complementary, palindrome or repeat sequences that are shared between the different target nucleic acid sequences can be used to shorten the overall length of the multifunctional siNA constructs (see for example FIGS. 18 and 19).

In a non-limiting example, three additional categories of additional multifunctional siNA designs are presented that allow a single siNA molecule to silence multiple targets. The first method utilizes linkers to join siNAs (or multifunctional siNAs) in a direct manner. This can allow the most potent siNAs to be joined without creating a long, continuous stretch of RNA that has potential to trigger an interferon response. The second method is a dendrimeric extension of the overlapping or the linked multifunctional design; or alternatively the organization of siNA in a supramolecular format. The third method uses helix lengths greater than 30 base pairs. Processing of these siNAs by Dicer will reveal new, active 5′ antisense ends. Therefore, the long siNAs can target the sites defined by the original 5′ ends and those defined by the new ends that are created by Dicer processing. When used in combination with traditional multifunctional siNAs (where the sense and antisense strands each define a target) the approach can be used for example to target 4 or more sites.

I. Tethered Bifunctional siNAs

The basic idea is a novel approach to the design of multifunctional siNAs in which two antisense siNA strands are annealed to a single sense strand. The sense strand oligonucleotide contains a linker (e.g., non-nucleotide linker as described herein) and two segments that anneal to the antisense siNA strands (see FIG. 22). The linkers can also optionally comprise nucleotide-based linkers. Several potential advantages and variations to this approach include, but are not limited to:

1. The two antisense siNAs are independent. Therefore, the choice of target sites is not constrained by a requirement for sequence conservation between two sites. Any two highly active siNAs can be combined to form a multifunctional siNA.
2. When used in combination with target sites having homology, siNAs that target a sequence present in two genes (e.g., different interleukin and/or interleukin receptor isoforms), the design can be used to target more than two sites. A single multifunctional siNA can be for example, used to target RNA of two different interleukin and/or interleukin receptor RNAs.
3. Multifunctional siNAs that use both the sense and antisense strands to target a gene can also be incorporated into a tethered multifunctional design. This leaves open the possibility of targeting 6 or more sites with a single complex.
4. It can be possible to anneal more than two antisense strand siNAs to a single tethered sense strand.
5. The design avoids long continuous stretches of dsRNA. Therefore, it is less likely to initiate an interferon response.
6. The linker (or modifications attached to it, such as conjugates described herein) can improve the pharmacokinetic properties of the complex or improve its incorporation into liposomes. Modifications introduced to the linker should not impact siNA activity to the same extent that they would if directly attached to the siNA (see for example FIGS. 27 and 28).
7. The sense strand can extend beyond the annealed antisense strands to provide additional sites for the attachment of conjugates.
8. The polarity of the complex can be switched such that both of the antisense 3′ ends are adjacent to the linker and the 5′ ends are distal to the linker or combination thereof.

Dendrimer and Supramolecular siNAs

In the dendrimer siNA approach, the synthesis of siNA is initiated by first synthesizing the dendrimer template followed by attaching various functional siNAs. Various constructs are depicted in FIG. 23. The number of functional siNAs that can be attached is only limited by the dimensions of the dendrimer used.

Supramolecular Approach to Multifunctional siNA

The supramolecular format simplifies the challenges of dendrimer synthesis. In this format, the siNA strands are synthesized by standard RNA chemistry, followed by annealing of various complementary strands. The individual strand synthesis contains an antisense sense sequence of one siNA at the 5′-end followed by a nucleic acid or synthetic linker, such as hexaethyleneglyol, which in turn is followed by sense strand of another siNA in 5′ to 3′ direction. Thus, the synthesis of siNA strands can be carried out in a standard 3′ to 5′ direction. Representative examples of trifunctional and tetrafunctional siNAs are depicted in FIG. 24. Based on a similar principle, higher functionality siNA constructs can be designed as long as efficient annealing of various strands is achieved.

Dicer Enabled Multifunctional siNA

Using bioinformatic analysis of multiple targets, stretches of identical sequences shared between differing target sequences can be identified ranging from about two to about fourteen nucleotides in length. These identical regions can be designed into extended siNA helixes (e.g., >30 base pairs) such that the processing by Dicer reveals a secondary functional 5′-antisense site (see for example FIG. 25). For example, when the first 17 nucleotides of a siNA antisense strand (e.g., 21 nucleotide strands in a duplex with 3′-TT overhangs) are complementary to a target RNA, robust silencing was observed at 25 nM. 80% silencing was observed with only 16 nucleotide complementarity in the same format.

Incorporation of this property into the designs of siNAs of about 30 to 40 or more base pairs results in additional multifunctional siNA constructs. The example in FIG. 25 illustrates how a 30 base-pair duplex can target three distinct sequences after processing by Dicer-RNaseIII; these sequences can be on the same mRNA or separate RNAs, such as viral and host factor messages, or multiple points along a given pathway (e.g., inflammatory cascades). Furthermore, a 40 base-pair duplex can combine a bifunctional design in tandem, to provide a single duplex targeting four target sequences. An even more extensive approach can include use of homologous sequences to enable five or six targets silenced for one multifunctional duplex. The example in FIG. 25 demonstrates how this can be achieved. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2′-OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30 mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs. Another non-limiting example is shown in FIG. 26. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Four targeting sequences are shown in four colors, blue, light-blue and red and orange. The required sequence identity overlapped is indicated by grey boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.

Example 12
Diagnostic Uses

The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates. siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).

In a specific example, siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules (i.e., those that cleave only wild-type forms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules (i.e., those that cleave only mutant forms of target RNA) are used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

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

It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying siNA molecules with improved RNAi activity.

The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

TABLE I

Interleukin and Interleukin receptor Accession Numbers

Interleukin Family

NM_000575

Homo sapiens interleukin 1, alpha (IL1A), mRNA

NM_000576

Homo sapiens interleukin 1, beta (IL1B), mRNA

NM_012275

Homo sapiens interleukin 1 family, member 5 (delta) (IL1F5), mRNA

NM_014440

Homo sapiens interleukin 1 family, member 6 (epsilon) (IL1F6), mRNA

NM_014439

Homo sapiens interleukin 1 family, member 7 (zeta) (IL1F7), mRNA

NM_014438

Homo sapiens interleukin 1 family, member 8 (eta) (IL1F8), mRNA

NM_019618

Homo sapiens interleukin 1 family, member 9 (IL1F9), mRNA

NM_032556

Homo sapiens interleukin 1 family, member 10 (theta) (IL1F10), mRNA

NM_000586

Homo sapiens interleukin 2 (IL2), mRNA

NM_000588

Homo sapiens interleukin 3 (colony-stimulating factor, multiple) (IL3),

mRNA

NM_000589

Homo sapiens interleukin 4 (IL4), mRNA

NM_000879

Homo sapiens interleukin 5 (colony-stimulating factor, eosinophil) (IL5),

mRNA

NM_000600

Homo sapiens interleukin 6 (interferon, beta 2) (IL6), mRNA

NM_000880

Homo sapiens interleukin 7 (IL7), mRNA

NM_000584

Homo sapiens interleukin 8 (IL8), mRNA

NM_000590

Homo sapiens interleukin 9 (IL9), mRNA

NM_000572

Homo sapiens interleukin 10 (IL10), mRNA

NM_000641

Homo sapiens interleukin 11 (IL11), mRNA

NM_000882

Homo sapiens interleukin 12A (natural killer cell stimulatory factor 1,

cytotoxic lymphocyte maturation factor 1, p35) (IL12A), mRNA

NM_002187

Homo sapiens interleukin 12B (natural killer cell stimulatory factor 2,

cytotoxic lymphocyte maturation factor 2, p40) (IL12B), mRNA

NM_002188

Homo sapiens interleukin 13 (IL13), mRNA

L15344

Homo sapiens interleukin 14 (IL14), mRNA

NM_000585

Homo sapiens interleukin 15 (IL15), mRNA

NM_004513

Homo sapiens interleukin 16 (lymphocyte chemoattractant factor) (IL16),

mRNA

NM_002190

Homo sapiens interleukin 17 (cytotoxic T-lymphocyte-associated serine

esterase 8) (IL17), mRNA

NM_014443

Homo sapiens interleukin 17B (IL17B), mRNA

NM_013278

Homo sapiens interleukin 17C (IL17C), mRNA

NM_138284

Homo sapiens interleukin 17D (IL17D), mRNA

NM_022789

Homo sapiens interleukin 17E (IL17E), mRNA

NM_052872

Homo sapiens interleukin 17F (IL17F), mRNA

NM_001562

Homo sapiens interleukin 18 (interferon-gamma-inducing factor) (IL18),

mRNA

NM_013371

Homo sapiens interleukin 19 (IL19), mRNA

NM_018724

Homo sapiens interleukin 20 (IL20), mRNA

NM_021803

Homo sapiens interleukin 21 (IL21 antisense), mRNA

NM_020525

Homo sapiens interleukin 22 (IL22), mRNA

NM_016584

Homo sapiens interleukin 23, alpha subunit p19 (IL23A), mRNA

NM_006850

Homo sapiens interleukin 24 (IL24), mRNA

NM_018402

Homo sapiens interleukin 26 (IL26), mRNA

AL365373

Homo sapiens interleukin 27 (IL27), mRNA

Interleukin Receptor Family

NM_000877

Homo sapiens interleukin 1 receptor, type I (IL1R1), mRNA

NM_004633

Homo sapiens interleukin 1 receptor, type II (IL1R2), mRNA

NM_016232

Homo sapiens interleukin 1 receptor-like 1 (IL1RL1), mRNA

NM_003856

Homo sapiens interleukin 1 receptor-like 1 (IL1RL1), mRNA

NM_003854

Homo sapiens interleukin 1 receptor-like 2 (IL1RL2), mRNA

NM_000417

Homo sapiens interleukin 2 receptor, alpha (IL2RA), mRNA

NM_000878

Homo sapiens interleukin 2 receptor, beta (IL2RB), mRNA

NM_000206

Homo sapiens interleukin 2 receptor, gamma (severe combined

immunodeficiency) (IL2RG), mRNA

NM_002183

Homo sapiens interleukin 3 receptor, alpha (low affinity) (IL3RA),

mRNA

NM_000418

Homo sapiens interleukin 4 receptor (IL4R), mRNA

NM_000564

Homo sapiens interleukin 5 receptor, alpha (IL5RA), mRNA

NM_000565

Homo sapiens interleukin 6 receptor (IL6R), mRNA

NM_002185

Homo sapiens interleukin 7 receptor (IL7R), mRNA

NM_000634

Homo sapiens interleukin 8 receptor, alpha (IL8RA), mRNA

NM_001557

Homo sapiens interleukin 8 receptor, beta (IL8RB), mRNA

NM_002186

Homo sapiens interleukin 9 receptor (IL9R), mRNA

NM_001558

Homo sapiens interleukin 10 receptor, alpha (IL10RA), mRNA

NM_000628

Homo sapiens interleukin 10 receptor, beta (IL10RB), mRNA

NM_004512

Homo sapiens interleukin 11 receptor, alpha (IL11RA), mRNA

NM_005535

Homo sapiens interleukin 12 receptor, beta 1 (IL12RB1), mRNA

NM_001559

Homo sapiens interleukin 12 receptor, beta 2 (IL12RB2), mRNA

NM_001560

Homo sapiens interleukin 13 receptor, alpha 1 (IL13RA1), mRNA

NM_000640

Homo sapiens interleukin 13 receptor, alpha 2 (IL13RA2), mRNA

NM_002189

Homo sapiens interleukin 15 receptor, alpha (IL15RA), mRNA

NM_014339

Homo sapiens interleukin 17 receptor (IL17R), mRNA

NM_032732

Homo sapiens interleukin 17 receptor C (IL-17RC), mRNA

NM_144640

Homo sapiens interleukin 17 receptor E (IL-17RE), mRNA

NM_018725

Homo sapiens interleukin 17B receptor (IL17BR), mRNA

NM_003855

Homo sapiens interleukin 18 receptor 1 (IL18R1), mRNA

NM_003853

Homo sapiens interleukin 18 receptor accessory protein (IL18RAP),

mRNA

NM_014432

Homo sapiens interleukin 20 receptor, alpha (IL20RA), mRNA

NM_021798

Homo sapiens interleukin 21 receptor (IL21 antisenseR), mRNA

NM_021258

Homo sapiens interleukin 22 receptor (IL22R), mRNA

NM_144701

Homo sapiens interleukin 23 receptor (IL23R), mRNA

Interleukin Associated Proteins

NM_004514

Homo sapiens interleukin enhancer binding factor 1 (ILF1), mRNA

NM_004515

Homo sapiens interleukin enhancer binding factor 2, 45 kD (ILF2), mRNA

NM_012218

Homo sapiens interleukin enhancer binding factor 3, 90 kD (ILF3), mRNA

NM_004516

Homo sapiens interleukin enhancer binding factor 3, 90 kD (ILF3), mRNA

NM_016123

Homo sapiens interleukin-1 receptor associated kinase 4 (IRAK4), mRNA

NM_001569

Homo sapiens interleukin-1 receptor-associated kinase 1 (IRAK1),

mRNA

NM_001570

Homo sapiens interleukin-1 receptor-associated kinase 2 (IRAK2),

mRNA

NM_007199

Homo sapiens interleukin-1 receptor-associated kinase 3 (IRAK3),

mRNA

NM_134470

Homo sapiens interleukin 1 receptor accessory protein (IL1RAP), mRNA

NM_002182

Homo sapiens interleukin 1 receptor accessory protein (IL1RAP), mRNA

NM_014271

Homo sapiens interleukin 1 receptor accessory protein-like 1

(IL1RAPL1), mRNA

NM_017416

Homo sapiens interleukin 1 receptor accessory protein-like 2

(IL1RAPL2), mRNA

NM_000577

Homo sapiens interleukin 1 receptor antagonist (IL1RN), mRNA

NM_002184

Homo sapiens interleukin 6 signal transducer (gp130, oncostatin M

receptor) (IL6ST), mRNA

NM_005699

Homo sapiens interleukin 18 binding protein (IL18BP), mRNA

TABLE II

Interleukin and Interleukin receptor siNA and Target Sequences

Seq

Seq

Seq

Pos
Seq
ID
UPos
Upper seq
ID
LPos
Lower seq
ID

IL2RG NM_000206

3
AGAGCAAGCGCCAUGUUGA
1
3
AGAGCAAGCGCCAUGUUGA
1
25
UCAACAUGGCGCUUGCUCU
82

21
AAGCCAUCAUUACCAUUCA
2
21
AAGCCAUCAUUACCAUUCA
2
43
UGAAUGGUAAUGAUGGCUU
83

39
ACAUCCCUCUUAUUCCUGC
3
39
ACAUCCCUCUUAUUCCUGC
3
61
GCAGGAAUAAGAGGGAUGU
84

57
CAGCUGCCCCUGCUGGGAG
4
57
CAGCUGCCCCUGCUGGGAG
4
79
CUCCCAGCAGGGGCAGCUG
85

75
GUGGGGCUGAACACGACAA
5
75
GUGGGGCUGAACACGACAA
5
97
UUGUCGUGUUCAGCCCCAC
86

93
AUUCUGACGCCCAAUGGGA
6
93
AUUCUGACGCCCAAUGGGA
6
115
UCCCAUUGGGCGUCAGAAU
87

111
AAUGAAGACACCACAGCUG
7
111
AAUGAAGACACCACAGCUG
7
133
CAGCUGUGGUGUCUUCAUU
88

129
GAUUUCUUCCUGACCACUA
8
129
GAUUUCUUCCUGACCACUA
8
151
UAGUGGUCAGGAAGAAAUC
89

147
AUGCCCACUGACUCCCUCA
9
147
AUGCCCACUGACUCCCUCA
9
169
UGAGGGAGUCAGUGGGCAU
90

165
AGUGUUUCCACUCUGCCCC
10
165
AGUGUUUCCACUCUGCCCC
10
187
GGGGCAGAGUGGAAACACU
91

183
CUCCCAGAGGUUCAGUGUU
11
183
CUCCCAGAGGUUCAGUGUU
11
205
AACACUGAACCUCUGGGAG
92

201
UUUGUGUUCAAUGUCGAGU
12
201
UUUGUGUUCAAUGUCGAGU
12
223
ACUCGACAUUGAACACAAA
93

219
UACAUGAAUUGCACUUGGA
13
219
UACAUGAAUUGCACUUGGA
13
241
UCCAAGUGCAAUUCAUGUA
94

237
AACAGCAGCUCUGAGCCCC
14
237
AACAGCAGCUCUGAGCCCC
14
259
GGGGCUCAGAGCUGCUGUU
95

255
CAGCCUACCAACCUCACUC
15
255
CAGCCUACCAACCUCACUC
15
277
GAGUGAGGUUGGUAGGCUG
96

273
CUGCAUUAUUGGUACAAGA
16
273
CUGCAUUAUUGGUACAAGA
16
295
UCUUGUACCAAUAAUGCAG
97

291
AACUCGGAUAAUGAUAAAG
17
291
AACUCGGAUAAUGAUAAAG
17
313
CUUUAUCAUUAUCCGAGUU
98

309
GUCCAGAAGUGCAGCCACU
18
309
GUCCAGAAGUGCAGCCACU
18
331
AGUGGCUGCACUUCUGGAC
99

327
UAUCUAUUCUCUGAAGAAA
19
327
UAUCUAUUCUCUGAAGAAA
19
349
UUUCUUCAGAGAAUAGAUA
100

345
AUCACUUCUGGCUGUCAGU
20
345
AUCACUUCUGGCUGUCAGU
20
367
ACUGACAGCCAGAAGUGAU
101

363
UUGCAAAAAAAGGAGAUCC
21
363
UUGCAAAAAAAGGAGAUCC
21
385
GGAUCUCCUUUUUUUGCAA
102

381
CACCUCUACCAAACAUUUG
22
381
CACCUCUACCAAACAUUUG
22
403
CAAAUGUUUGGUAGAGGUG
103

399
GUUGUUCAGCUCCAGGACC
23
399
GUUGUUCAGCUCCAGGACC
23
421
GGUCCUGGAGCUGAACAAC
104

417
CCACGGGAACCCAGGAGAC
24
417
CCACGGGAACCCAGGAGAC
24
439
GUCUCCUGGGUUCCCGUGG
105

435
CAGGCCACACAGAUGCUAA
25
435
CAGGCCACACAGAUGCUAA
25
457
UUAGCAUCUGUGUGGCCUG
106

453
AAACUGCAGAAUCUGGUGA
26
453
AAACUGCAGAAUCUGGUGA
26
475
UCACCAGAUUCUGCAGUUU
107

471
AUCCCCUGGGCUCCAGAGA
27
471
AUCCCCUGGGCUCCAGAGA
27
493
UCUCUGGAGCCCAGGGGAU
108

489
AACCUAACACUUCACAAAC
28
489
AACCUAACACUUCACAAAC
28
511
GUUUGUGAAGUGUUAGGUU
109

507
CUGAGUGAAUCCCAGCUAG
29
507
CUGAGUGAAUCCCAGCUAG
29
529
CUAGCUGGGAUUCACUCAG
110

525
GAACUGAACUGGAACAACA
30
525
GAACUGAACUGGAACAACA
30
547
UGUUGUUCCAGUUCAGUUC
111

543
AGAUUCUUGAACCACUGUU
31
543
AGAUUCUUGAACCACUGUU
31
565
AACAGUGGUUCAAGAAUCU
112

561
UUGGAGCACUUGGUGCAGU
32
561
UUGGAGCACUUGGUGCAGU
32
583
ACUGCACCAAGUGCUCCAA
113

579
UACCGGACUGACUGGGACC
33
579
UACCGGACUGACUGGGACC
33
601
GGUCCCAGUCAGUCCGGUA
114

597
CACAGCUGGACUGAACAAU
34
597
CACAGCUGGACUGAACAAU
34
619
AUUGUUCAGUCCAGCUGUG
115

615
UCAGUGGAUUAUAGACAUA
35
615
UCAGUGGAUUAUAGACAUA
35
637
UAUGUCUAUAAUCCACUGA
116

633
AAGUUCUCCUUGCCUAGUG
36
633
AAGUUCUCCUUGCCUAGUG
36
655
CACUAGGCAAGGAGAACUU
117

651
GUGGAUGGGCAGAAACGCU
37
651
GUGGAUGGGCAGAAACGCU
37
673
AGCGUUUCUGCCCAUCCAC
118

669
UACACGUUUCGUGUUCGGA
38
669
UACACGUUUCGUGUUCGGA
38
691
UCCGAACACGAAACGUGUA
119

687
AGCCGCUUUAACCCACUCU
39
687
AGCCGCUUUAACCCACUCU
39
709
AGAGUGGGUUAAAGCGGCU
120

705
UGUGGAAGUGCUCAGCAUU
40
705
UGUGGAAGUGCUCAGCAUU
40
727
AAUGCUGAGCACUUCCACA
121

723
UGGAGUGAAUGGAGCCACC
41
723
UGGAGUGAAUGGAGCCACC
41
745
GGUGGCUCCAUUCACUCCA
122

741
CCAAUCCACUGGGGGAGCA
42
741
CCAAUCCACUGGGGGAGCA
42
763
UGCUCCCCCAGUGGAUUGG
123

759
AAUACUUCAAAAGAGAAUC
43
759
AAUACUUCAAAAGAGAAUC
43
781
GAUUCUCUUUUGAAGUAUU
124

777
CCUUUCCUGUUUGCAUUGG
44
777
CCUUUCCUGUUUGCAUUGG
44
799
CCAAUGCAAACAGGAAAGG
125

795
GAAGCCGUGGUUAUCUCUG
45
795
GAAGCCGUGGUUAUCUCUG
45
817
CAGAGAUAACCACGGCUUC
126

813
GUUGGCUCCAUGGGAUUGA
46
813
GUUGGCUCCAUGGGAUUGA
46
835
UCAAUCCCAUGGAGCCAAC
127

831
AUUAUCAGCCUUCUCUGUG
47
831
AUUAUCAGCCUUCUCUGUG
47
853
CACAGAGAAGGCUGAUAAU
128

849
GUGUAUUUCUGGCUGGAAC
48
849
GUGUAUUUCUGGCUGGAAC
48
871
GUUCCAGCCAGAAAUACAC
129

867
CGGACGAUGCCCCGAAUUC
49
867
CGGACGAUGCCCCGAAUUC
49
889
GAAUUCGGGGCAUCGUCCG
130

885
CCCACCCUGAAGAACCUAG
50
885
CCCACCCUGAAGAACCUAG
50
907
CUAGGUUCUUCAGGGUGGG
131

903
GAGGAUCUUGUUACUGAAU
51
903
GAGGAUCUUGUUACUGAAU
51
925
AUUCAGUAACAAGAUCCUC
132

921
UACCACGGGAACUUUUCGG
52
921
UACCACGGGAACUUUUCGG
52
943
CCGAAAAGUUCCCGUGGUA
133

939
GCCUGGAGUGGUGUGUCUA
53
939
GCCUGGAGUGGUGUGUCUA
53
961
UAGACACACCACUCCAGGC
134

957
AAGGGACUGGCUGAGAGUC
54
957
AAGGGACUGGCUGAGAGUC
54
979
GACUCUCAGCCAGUCCCUU
135

975
CUGCAGCCAGACUACAGUG
55
975
CUGCAGCCAGACUACAGUG
55
997
CACUGUAGUCUGGCUGCAG
136

993
GAACGACUCUGCCUCGUCA
56
993
GAACGACUCUGCCUCGUCA
56
1015
UGACGAGGCAGAGUCGUUC
137

1011
AGUGAGAUUCCCCCAAAAG
57
1011
AGUGAGAUUCCCCCAAAAG
57
1033
CUUUUGGGGGAAUCUCACU
138

1029
GGAGGGGCCCUUGGGGAGG
58
1029
GGAGGGGCCCUUGGGGAGG
58
1051
CCUCCCCAAGGGCCCCUCC
139

1047
GGGCCUGGGGCCUCCCCAU
59
1047
GGGCCUGGGGCCUCCCCAU
59
1069
AUGGGGAGGCCCCAGGCCC
140

1065
UGCAACCAGCAUAGCCCCU
60
1065
UGCAACCAGCAUAGCCCCU
60
1087
AGGGGCUAUGCUGGUUGCA
141

1083
UACUGGGCCCCCCCAUGUU
61
1083
UACUGGGCCCCCCCAUGUU
61
1105
AACAUGGGGGGGCCCAGUA
142

1101
UACACCCUAAAGCCUGAAA
62
1101
UACACCCUAAAGCCUGAAA
62
1123
UUUCAGGCUUUAGGGUGUA
143

1119
ACCUGAACCCCAAUCCUCU
63
1119
ACCUGAACCCCAAUCCUCU
63
1141
AGAGGAUUGGGGUUCAGGU
144

1137
UGACAGAAGAACCCCAGGG
64
1137
UGACAGAAGAACCCCAGGG
64
1159
CCCUGGGGUUCUUCUGUCA
145

1155
GUCCUGUAGCCCUAAGUGG
65
1155
GUCCUGUAGCCCUAAGUGG
65
1177
CCACUUAGGGCUACAGGAC
146

1173
GUACUAACUUUCCUUCAUU
66
1173
GUACUAACUUUCCUUCAUU
66
1195
AAUGAAGGAAAGUUAGUAC
147

1191
UCAACCCACCUGCGUCUCA
67
1191
UCAACCCACCUGCGUCUCA
67
1213
UGAGACGCAGGUGGGUUGA
148

1209
AUACUCACCUCACCCCACU
68
1209
AUACUCACCUCACCCCACU
68
1231
AGUGGGGUGAGGUGAGUAU
149

1227
UGUGGCUGAUUUGGAAUUU
69
1227
UGUGGCUGAUUUGGAAUUU
69
1249
AAAUUCCAAAUCAGCCACA
150

1245
UUGUGCCCCCAUGUAAGCA
70
1245
UUGUGCCCCCAUGUAAGCA
70
1267
UGCUUACAUGGGGGCACAA
151

1263
ACCCCUUCAUUUGGCAUUC
71
1263
ACCCCUUCAUUUGGCAUUC
71
1285
GAAUGCCAAAUGAAGGGGU
152

1281
CCCCACUUGAGAAUUACCC
72
1281
CCCCACUUGAGAAUUACCC
72
1303
GGGUAAUUCUCAAGUGGGG
153

1299
CUUUUGCCCCGAACAUGUU
73
1299
CUUUUGCCCCGAACAUGUU
73
1321
AACAUGUUCGGGGCAAAAG
154

1317
UUUUCUUCUCCCUCAGUCU
74
1317
UUUUCUUCUCCCUCAGUCU
74
1339
AGACUGAGGGAGAAGAAAA
155

1335
UGGCCCUUCCUUUUCGCAG
75
1335
UGGCCCUUCCUUUUCGCAG
75
1357
CUGCGAAAAGGAAGGGCCA
156

1353
GGAUUCUUCCUCCCUCCCU
76
1353
GGAUUCUUCCUCCCUCCCU
76
1375
AGGGAGGGAGGAAGAAUCC
157

1371
UCUUUCCCUCCCUUCCUCU
77
1371
UCUUUCCCUCCCUUCCUCU
77
1393
AGAGGAAGGGAGGGAAAGA
158

1389
UUUCCAUCUACCCUCCGAU
78
1389
UUUCCAUCUACCCUCCGAU
78
1411
AUCGGAGGGUAGAUGGAAA
159

1407
UUGUUCCUGAACCGAUGAG
79
1407
UUGUUCCUGAACCGAUGAG
79
1429
CUCAUCGGUUCAGGAACAA
160

1425
GAAAUAAAGUUUCUGUUGA
80
1425
GAAAUAAAGUUUCUGUUGA
80
1447
UCAACAGAAACUUUAUUUC
161

1431
AAGUUUCUGUUGAUAAUCA
81
1431
AAGUUUCUGUUGAUAAUCA
81
1453
UGAUUAUCAACAGAAACUU
162

IL4 NM_000589

3
CUAUGCAAAGCAAAAAGCC
163
3
CUAUGCAAAGCAAAAAGCC
163
25
GGCUUUUUGCUUUGCAUAG
214

21
CAGCAGCAGCCCCAAGCUG
164
21
CAGCAGCAGCCCCAAGCUG
164
43
CAGCUUGGGGCUGCUGCUG
215

39
GAUAAGAUUAAUCUAAAGA
165
39
GAUAAGAUUAAUCUAAAGA
165
61
UCUUUAGAUUAAUCUUAUC
216

57
AGCAAAUUAUGGUGUAAUU
166
57
AGCAAAUUAUGGUGUAAUU
166
79
AAUUACACCAUAAUUUGCU
217

75
UUCCUAUGCUGAAACUUUG
167
75
UUCCUAUGCUGAAACUUUG
167
97
CAAAGUUUCAGCAUAGGAA
218

93
GUAGUUAAUUUUUUAAAAA
168
93
GUAGUUAAUUUUUUAAAAA
168
115
UUUUUAAAAAAUUAACUAC
219

111
AGGUUUCAUUUUCCUAUUG
169
111
AGGUUUCAUUUUCCUAUUG
169
133
CAAUAGGAAAAUGAAACCU
220

129
GGUCUGAUUUCACAGGAAC
170
129
GGUCUGAUUUCACAGGAAC
170
151
GUUCCUGUGAAAUCAGACC
221

147
CAUUUUACCUGUUUGUGAG
171
147
CAUUUUACCUGUUUGUGAG
171
169
CUCACAAACAGGUAAAAUG
222

165
GGCAUUUUUUCUCCUGGAA
172
165
GGCAUUUUUUCUCCUGGAA
172
187
UUCCAGGAGAAAAAAUGCC
223

183
AGAGAGGUGCUGAUUGGCC
173
183
AGAGAGGUGCUGAUUGGCC
173
205
GGCCAAUCAGCACCUCUCU
224

201
CCCAAGUGACUGACAAUCU
174
201
CCCAAGUGACUGACAAUCU
174
223
AGAUUGUCAGUCACUUGGG
225

219
UGGUGUAACGAAAAUUUCC
175
219
UGGUGUAACGAAAAUUUCC
175
241
GGAAAUUUUCGUUACACCA
226

237
CAAUGUAAACUCAUUUUCC
176
237
CAAUGUAAACUCAUUUUCC
176
259
GGAAAAUGAGUUUACAUUG
227

255
CCUCGGUUUCAGCAAUUUU
177
255
CCUCGGUUUCAGCAAUUUU
177
277
AAAAUUGCUGAAACCGAGG
228

273
UAAAUCUAUAUAUAGAGAU
178
273
UAAAUCUAUAUAUAGAGAU
178
295
AUCUCUAUAUAUAGAUUUA
229

291
UAUCUUUGUCAGCAUUGCA
179
291
UAUCUUUGUCAGCAUUGCA
179
313
UGCAAUGCUGACAAAGAUA
230

309
AUCGUUAGCUUCUCCUGAU
180
309
AUCGUUAGCUUCUCCUGAU
180
331
AUCAGGAGAAGCUAACGAU
231

327
UAAACUAAUUGCCUCACAU
181
327
UAAACUAAUUGCCUCACAU
181
349
AUGUGAGGCAAUUAGUUUA
232

345
UUGUCACUGCAAAUCGACA
182
345
UUGUCACUGCAAAUCGACA
182
367
UGUCGAUUUGCAGUGACAA
233

363
ACCUAUUAAUGGGUCUCAC
183
363
ACCUAUUAAUGGGUCUCAC
183
385
GUGAGACCCAUUAAUAGGU
234

381
CCUCCCAACUGCUUCCCCC
184
381
CCUCCCAACUGCUUCCCCC
184
403
GGGGGAAGCAGUUGGGAGG
235

399
CUCUGUUCUUCCUGCUAGC
185
399
CUCUGUUCUUCCUGCUAGC
185
421
GCUAGCAGGAAGAACAGAG
236

417
CAUGUGCCGGCAACUUUGU
186
417
CAUGUGCCGGCAACUUUGU
186
439
ACAAAGUUGCCGGCACAUG
237

435
UCCACGGACACAAGUGCGA
187
435
UCCACGGACACAAGUGCGA
187
457
UCGCACUUGUGUCCGUGGA
238

453
AUAUCACCUUACAGGAGAU
188
453
AUAUCACCUUACAGGAGAU
188
475
AUCUCCUGUAAGGUGAUAU
239

471
UCAUCAAAACUUUGAACAG
189
471
UCAUCAAAACUUUGAACAG
189
493
CUGUUCAAAGUUUUGAUGA
240

489
GCCUCACAGAGCAGAAGAC
190
489
GCCUCACAGAGCAGAAGAC
190
511
GUCUUCUGCUCUGUGAGGC
241

507
CUCUGUGCACCGAGUUGAC
191
507
CUCUGUGCACCGAGUUGAC
191
529
GUCAACUCGGUGCACAGAG
242

525
CCGUAACAGACAUCUUUGC
192
525
CCGUAACAGACAUCUUUGC
192
547
GCAAAGAUGUCUGUUACGG
243

543
CUGCCUCCAAGAACACAAC
193
543
CUGCCUCCAAGAACACAAC
193
565
GUUGUGUUCUUGGAGGCAG
244

561
CUGAGAAGGAAACCUUCUG
194
561
CUGAGAAGGAAACCUUCUG
194
583
CAGAAGGUUUCCUUCUCAG
245

579
GCAGGGCUGCGACUGUGCU
195
579
GCAGGGCUGCGACUGUGCU
195
601
AGCACAGUCGCAGCCCUGC
246

597
UCCGGCAGUUCUACAGCCA
196
597
UCCGGCAGUUCUACAGCCA
196
619
UGGCUGUAGAACUGCCGGA
247

615
ACCAUGAGAAGGACACUCG
197
615
ACCAUGAGAAGGACACUCG
197
637
CGAGUGUCCUUCUCAUGGU
248

633
GCUGCCUGGGUGCGACUGC
198
633
GCUGCCUGGGUGCGACUGC
198
655
GCAGUCGCACCCAGGCAGC
249

651
CACAGCAGUUCCACAGGCA
199
651
CACAGCAGUUCCACAGGCA
199
673
UGCCUGUGGAACUGCUGUG
250

669
ACAAGCAGCUGAUCCGAUU
200
669
ACAAGCAGCUGAUCCGAUU
200
691
AAUCGGAUCAGCUGCUUGU
251

687
UCCUGAAACGGCUCGACAG
201
687
UCCUGAAACGGCUCGACAG
201
709
CUGUCGAGCCGUUUCAGGA
252

705
GGAACCUCUGGGGCCUGGC
202
705
GGAACCUCUGGGGCCUGGC
202
727
GCCAGGCCCCAGAGGUUCC
253

723
CGGGCUUGAAUUCCUGUCC
203
723
CGGGCUUGAAUUCCUGUCC
203
745
GGACAGGAAUUCAAGCCCG
254

741
CUGUGAAGGAAGCCAACCA
204
741
CUGUGAAGGAAGCCAACCA
204
763
UGGUUGGCUUCCUUCACAG
255

759
AGAGUACGUUGGAAAACUU
205
759
AGAGUACGUUGGAAAACUU
205
781
AAGUUUUCCAACGUACUCU
256

777
UCUUGGAAAGGCUAAAGAC
206
777
UCUUGGAAAGGCUAAAGAC
206
799
GUCUUUAGCCUUUCCAAGA
257

795
CGAUCAUGAGAGAGAAAUA
207
795
CGAUCAUGAGAGAGAAAUA
207
817
UAUUUCUCUCUCAUGAUCG
258

813
AUUCAAAGUGUUCGAGCUG
208
813
AUUCAAAGUGUUCGAGCUG
208
835
CAGCUCGAACACUUUGAAU
259

831
GAAUAUUUUAAUUUAUGAG
209
831
GAAUAUUUUAAUUUAUGAG
209
853
CUCAUAAAUUAAAAUAUUC
260

849
GUUUUUGAUAGCUUUAUUU
210
849
GUUUUUGAUAGCUUUAUUU
210
871
AAAUAAAGCUAUCAAAAAC
261

867
UUUUAAGUAUUUAUAUAUU
211
867
UUUUAAGUAUUUAUAUAUU
211
889
AAUAUAUAAAUACUUAAAA
262

885
UUAUAACUCAUCAUAAAAU
212
885
UUAUAACUCAUCAUAAAAU
212
907
AUUUUAUGAUGAGUUAUAA
263

901
AAUAAAGUAUAUAUAGAAU
213
901
AAUAAAGUAUAUAUAGAAU
213
923
AUUCUAUAUAUACUUUAUU
264

IL4R NM_000418

3
CGAAUGGAGCAGGGGCGCG
265
3
CGAAUGGAGCAGGGGCGCG
265
25
CGCGCCCCUGCUCCAUUCG
465

21
GCAGAUAAUUAAAGAUUUA
266
21
GCAGAUAAUUAAAGAUUUA
266
43
UAAAUCUUUAAUUAUCUGC
466

39
ACACACAGCUGGAAGAAAU
267
39
ACACACAGCUGGAAGAAAU
267
61
AUUUCUUCCAGCUGUGUGU
467

57
UCAUAGAGAAGCCGGGCGU
268
57
UCAUAGAGAAGCCGGGCGU
268
79
ACGCCCGGCUUCUCUAUGA
468

75
UGGUGGCUCAUGCCUAUAA
269
75
UGGUGGCUCAUGCCUAUAA
269
97
UUAUAGGCAUGAGCCACCA
469

93
AUCCCAGCACUUUUGGAGG
270
93
AUCCCAGCACUUUUGGAGG
270
115
CCUCCAAAAGUGCUGGGAU
470

111
GCUGAGGCGGGCAGAUCAC
271
111
GCUGAGGCGGGCAGAUCAC
271
133
GUGAUCUGCCCGCCUCAGC
471

129
CUUGAGAUCAGGAGUUCGA
272
129
CUUGAGAUCAGGAGUUCGA
272
151
UCGAACUCCUGAUCUCAAG
472

147
AGACCAGCCUGGUGCCUUG
273
147
AGACCAGCCUGGUGCCUUG
273
169
CAAGGCACCAGGCUGGUCU
473

165
GGCAUCUCCCAAUGGGGUG
274
165
GGCAUCUCCCAAUGGGGUG
274
187
CACCCCAUUGGGAGAUGCC
474

183
GGCUUUGCUCUGGGCUCCU
275
183
GGCUUUGCUCUGGGCUCCU
275
205
AGGAGCCCAGAGCAAAGCC
475

201
UGUUCCCUGUGAGCUGCCU
276
201
UGUUCCCUGUGAGCUGCCU
276
223
AGGCAGCUCACAGGGAACA
476

219
UGGUCCUGCUGCAGGUGGC
277
219
UGGUCCUGCUGCAGGUGGC
277
241
GCCACCUGCAGCAGGACCA
477

237
CAAGCUCUGGGAACAUGAA
278
237
CAAGCUCUGGGAACAUGAA
278
259
UUCAUGUUCCCAGAGCUUG
478

255
AGGUCUUGCAGGAGCCCAC
279
255
AGGUCUUGCAGGAGCCCAC
279
277
GUGGGCUCCUGCAAGACCU
479

273
CCUGCGUCUCCGACUACAU
280
273
CCUGCGUCUCCGACUACAU
280
295
AUGUAGUCGGAGACGCAGG
480

291
UGAGCAUCUCUACUUGCGA
281
291
UGAGCAUCUCUACUUGCGA
281
313
UCGCAAGUAGAGAUGCUCA
481

309
AGUGGAAGAUGAAUGGUCC
282
309
AGUGGAAGAUGAAUGGUCC
282
331
GGACCAUUCAUCUUCCACU
482

327
CCACCAAUUGCAGCACCGA
283
327
CCACCAAUUGCAGCACCGA
283
349
UCGGUGCUGCAAUUGGUGG
483

345
AGCUCCGCCUGUUGUACCA
284
345
AGCUCCGCCUGUUGUACCA
284
367
UGGUACAACAGGCGGAGCU
484

363
AGCUGGUUUUUCUGCUCUC
285
363
AGCUGGUUUUUCUGCUCUC
285
385
GAGAGCAGAAAAACCAGCU
485

381
CCGAAGCCCACACGUGUAU
286
381
CCGAAGCCCACACGUGUAU
286
403
AUACACGUGUGGGCUUCGG
486

399
UCCCUGAGAACAACGGAGG
287
399
UCCCUGAGAACAACGGAGG
287
421
CCUCCGUUGUUCUCAGGGA
487

417
GCGCGGGGUGCGUGUGCCA
288
417
GCGCGGGGUGCGUGUGCCA
288
439
UGGCACACGCACCCCGCGC
488

435
ACCUGCUCAUGGAUGACGU
289
435
ACCUGCUCAUGGAUGACGU
289
457
ACGUCAUCCAUGAGCAGGU
489

453
UGGUCAGUGCGGAUAACUA
290
453
UGGUCAGUGCGGAUAACUA
290
475
UAGUUAUCCGCACUGACCA
490

471
AUACACUGGACCUGUGGGC
291
471
AUACACUGGACCUGUGGGC
291
493
GCCCACAGGUCCAGUGUAU
491

489
CUGGGCAGCAGCUGCUGUG
292
489
CUGGGCAGCAGCUGCUGUG
292
511
CACAGCAGCUGCUGCCCAG
492

507
GGAAGGGCUCCUUCAAGCC
293
507
GGAAGGGCUCCUUCAAGCC
293
529
GGCUUGAAGGAGCCCUUCC
493

525
CCAGCGAGCAUGUGAAACC
294
525
CCAGCGAGCAUGUGAAACC
294
547
GGUUUCACAUGCUCGCUGG
494

543
CCAGGGCCCCAGGAAACCU
295
543
CCAGGGCCCCAGGAAACCU
295
565
AGGUUUCCUGGGGCCCUGG
495

561
UGACAGUUCACACCAAUGU
296
561
UGACAGUUCACACCAAUGU
296
583
ACAUUGGUGUGAACUGUCA
496

579
UCUCCGACACUCUGCUGCU
297
579
UCUCCGACACUCUGCUGCU
297
601
AGCAGCAGAGUGUCGGAGA
497

597
UGACCUGGAGCAACCCGUA
298
597
UGACCUGGAGCAACCCGUA
298
619
UACGGGUUGCUCCAGGUCA
498

615
AUCCCCCUGACAAUUACCU
299
615
AUCCCCCUGACAAUUACCU
299
637
AGGUAAUUGUCAGGGGGAU
499

633
UGUAUAAUCAUCUCACCUA
300
633
UGUAUAAUCAUCUCACCUA
300
655
UAGGUGAGAUGAUUAUACA
500

651
AUGCAGUCAACAUUUGGAG
301
651
AUGCAGUCAACAUUUGGAG
301
673
CUCCAAAUGUUGACUGCAU
501

669
GUGAAAACGACCCGGCAGA
302
669
GUGAAAACGACCCGGCAGA
302
691
UCUGCCGGGUCGUUUUCAC
502

687
AUUUCAGAAUCUAUAACGU
303
687
AUUUCAGAAUCUAUAACGU
303
709
ACGUUAUAGAUUCUGAAAU
503

705
UGACCUACCUAGAACCCUC
304
705
UGACCUACCUAGAACCCUC
304
727
GAGGGUUCUAGGUAGGUCA
504

723
CCCUCCGCAUCGCAGCCAG
305
723
CCCUCCGCAUCGCAGCCAG
305
745
CUGGCUGCGAUGCGGAGGG
505

741
GCACCCUGAAGUCUGGGAU
306
741
GCACCCUGAAGUCUGGGAU
306
763
AUCCCAGACUUCAGGGUGC
506

759
UUUCCUACAGGGCACGGGU
307
759
UUUCCUACAGGGCACGGGU
307
781
ACCCGUGCCCUGUAGGAAA
507

777
UGAGGGCCUGGGCUCAGUG
308
777
UGAGGGCCUGGGCUCAGUG
308
799
CACUGAGCCCAGGCCCUCA
508

795
GCUAUAACACCACCUGGAG
309
795
GCUAUAACACCACCUGGAG
309
817
CUCCAGGUGGUGUUAUAGC
509

813
GUGAGUGGAGCCCCAGCAC
310
813
GUGAGUGGAGCCCCAGCAC
310
835
GUGCUGGGGCUCCACUCAC
510

831
CCAAGUGGCACAACUCCUA
311
831
CCAAGUGGCACAACUCCUA
311
853
UAGGAGUUGUGCCACUUGG
511

849
ACAGGGAGCCCUUCGAGCA
312
849
ACAGGGAGCCCUUCGAGCA
312
871
UGCUCGAAGGGCUCCCUGU
512

867
AGCACCUCCUGCUGGGCGU
313
867
AGCACCUCCUGCUGGGCGU
313
889
ACGCCCAGCAGGAGGUGCU
513

885
UCAGCGUUUCCUGCAUUGU
314
885
UCAGCGUUUCCUGCAUUGU
314
907
ACAAUGCAGGAAACGCUGA
514

903
UCAUCCUGGCCGUCUGCCU
315
903
UCAUCCUGGCCGUCUGCCU
315
925
AGGCAGACGGCCAGGAUGA
515

921
UGUUGUGCUAUGUCAGCAU
316
921
UGUUGUGCUAUGUCAGCAU
316
943
AUGCUGACAUAGCACAACA
516

939
UCACCAAGAUUAAGAAAGA
317
939
UCACCAAGAUUAAGAAAGA
317
961
UCUUUCUUAAUCUUGGUGA
517

957
AAUGGUGGGAUCAGAUUCC
318
957
AAUGGUGGGAUCAGAUUCC
318
979
GGAAUCUGAUCCCACCAUU
518

975
CCAACCCAGCCCGCAGCCG
319
975
CCAACCCAGCCCGCAGCCG
319
997
CGGCUGCGGGCUGGGUUGG
519

993
GCCUCGUGGCUAUAAUAAU
320
993
GCCUCGUGGCUAUAAUAAU
320
1015
AUUAUUAUAGCCACGAGGC
520

1011
UCCAGGAUGCUCAGGGGUC
321
1011
UCCAGGAUGCUCAGGGGUC
321
1033
GACCCCUGAGCAUCCUGGA
521

1029
CACAGUGGGAGAAGCGGUC
322
1029
CACAGUGGGAGAAGCGGUC
322
1051
GACCGCUUCUCCCACUGUG
522

1047
CCCGAGGCCAGGAACCAGC
323
1047
CCCGAGGCCAGGAACCAGC
323
1069
GCUGGUUCCUGGCCUCGGG
523

1065
CCAAGUGCCCACACUGGAA
324
1065
CCAAGUGCCCACACUGGAA
324
1087
UUCCAGUGUGGGCACUUGG
524

1083
AGAAUUGUCUUACCAAGCU
325
1083
AGAAUUGUCUUACCAAGCU
325
1105
AGCUUGGUAAGACAAUUCU
525

1101
UCUUGCCCUGUUUUCUGGA
326
1101
UCUUGCCCUGUUUUCUGGA
326
1123
UCCAGAAAACAGGGCAAGA
526

1119
AGCACAACAUGAAAAGGGA
327
1119
AGCACAACAUGAAAAGGGA
327
1141
UCCCUUUUCAUGUUGUGCU
527

1137
AUGAAGAUCCUCACAAGGC
328
1137
AUGAAGAUCCUCACAAGGC
328
1159
GCCUUGUGAGGAUCUUCAU
528

1155
CUGCCAAAGAGAUGCCUUU
329
1155
CUGCCAAAGAGAUGCCUUU
329
1177
AAAGGCAUCUCUUUGGCAG
529

1173
UCCAGGGCUCUGGAAAAUC
330
1173
UCCAGGGCUCUGGAAAAUC
330
1195
GAUUUUCCAGAGCCCUGGA
530

1191
CAGCAUGGUGCCCAGUGGA
331
1191
CAGCAUGGUGCCCAGUGGA
331
1213
UCCACUGGGCACCAUGCUG
531

1209
AGAUCAGCAAGACAGUCCU
332
1209
AGAUCAGCAAGACAGUCCU
332
1231
AGGACUGUCUUGCUGAUCU
532

1227
UCUGGCCAGAGAGCAUCAG
333
1227
UCUGGCCAGAGAGCAUCAG
333
1249
CUGAUGCUCUCUGGCCAGA
533

1245
GCGUGGUGCGAUGUGUGGA
334
1245
GCGUGGUGCGAUGUGUGGA
334
1267
UCCACACAUCGCACCACGC
534

1263
AGUUGUUUGAGGCCCCGGU
335
1263
AGUUGUUUGAGGCCCCGGU
335
1285
ACCGGGGCCUCAAACAACU
535

1281
UGGAGUGUGAGGAGGAGGA
336
1281
UGGAGUGUGAGGAGGAGGA
336
1303
UCCUCCUCCUCACACUCCA
536

1299
AGGAGGUAGAGGAAGAAAA
337
1299
AGGAGGUAGAGGAAGAAAA
337
1321
UUUUCUUCCUCUACCUCCU
537

1317
AAGGGAGCUUCUGUGCAUC
338
1317
AAGGGAGCUUCUGUGCAUC
338
1339
GAUGCACAGAAGCUCCCUU
538

1335
CGCCUGAGAGCAGCAGGGA
339
1335
CGCCUGAGAGCAGCAGGGA
339
1357
UCCCUGCUGCUCUCAGGCG
539

1353
AUGACUUCCAGGAGGGAAG
340
1353
AUGACUUCCAGGAGGGAAG
340
1375
CUUCCCUCCUGGAAGUCAU
540

1371
GGGAGGGCAUUGUGGCCCG
341
1371
GGGAGGGCAUUGUGGCCCG
341
1393
CGGGCCACAAUGCCCUCCC
541

1389
GGCUAACAGAGAGCCUGUU
342
1389
GGCUAACAGAGAGCCUGUU
342
1411
AACAGGCUCUCUGUUAGCC
542

1407
UCCUGGACCUGCUCGGAGA
343
1407
UCCUGGACCUGCUCGGAGA
343
1429
UCUCCGAGCAGGUCCAGGA
543

1425
AGGAGAAUGGGGGCUUUUG
344
1425
AGGAGAAUGGGGGCUUUUG
344
1447
CAAAAGCCCCCAUUCUCCU
544

1443
GCCAGCAGGACAUGGGGGA
345
1443
GCCAGCAGGACAUGGGGGA
345
1465
UCCCCCAUGUCCUGCUGGC
545

1461
AGUCAUGCCUUCUUCCACC
346
1461
AGUCAUGCCUUCUUCCACC
346
1483
GGUGGAAGAAGGCAUGACU
546

1479
CUUCGGGAAGUACGAGUGC
347
1479
CUUCGGGAAGUACGAGUGC
347
1501
GCACUCGUACUUCCCGAAG
547

1497
CUCACAUGCCCUGGGAUGA
348
1497
CUCACAUGCCCUGGGAUGA
348
1519
UCAUCCCAGGGCAUGUGAG
548

1515
AGUUCCCAAGUGCAGGGCC
349
1515
AGUUCCCAAGUGCAGGGCC
349
1537
GGCCCUGCACUUGGGAACU
549

1533
CCAAGGAGGCACCUCCCUG
350
1533
CCAAGGAGGCACCUCCCUG
350
1555
CAGGGAGGUGCCUCCUUGG
550

1551
GGGGCAAGGAGCAGCCUCU
351
1551
GGGGCAAGGAGCAGCCUCU
351
1573
AGAGGCUGCUCCUUGCCCC
551

1569
UCCACCUGGAGCCAAGUCC
352
1569
UCCACCUGGAGCCAAGUCC
352
1591
GGACUUGGCUCCAGGUGGA
552

1587
CUCCUGCCAGCCCGACCCA
353
1587
CUCCUGCCAGCCCGACCCA
353
1609
UGGGUCGGGCUGGCAGGAG
553

1605
AGAGUCCAGACAACCUGAC
354
1605
AGAGUCCAGACAACCUGAC
354
1627
GUCAGGUUGUCUGGACUCU
554

1623
CUUGCACAGAGACGCCCCU
355
1623
CUUGCACAGAGACGCCCCU
355
1645
AGGGGCGUCUCUGUGCAAG
555

1641
UCGUCAUCGCAGGCAACCC
356
1641
UCGUCAUCGCAGGCAACCC
356
1663
GGGUUGCCUGCGAUGACGA
556

1659
CUGCUUACCGCAGCUUCAG
357
1659
CUGCUUACCGCAGCUUCAG
357
1681
CUGAAGCUGCGGUAAGCAG
557

1677
GCAACUCCCUGAGCCAGUC
358
1677
GCAACUCCCUGAGCCAGUC
358
1699
GACUGGCUCAGGGAGUUGC
558

1695
CACCGUGUCCCAGAGAGCU
359
1695
CACCGUGUCCCAGAGAGCU
359
1717
AGCUCUCUGGGACACGGUG
559

1713
UGGGUCCAGACCCACUGCU
360
1713
UGGGUCCAGACCCACUGCU
360
1735
AGCAGUGGGUCUGGACCCA
560

1731
UGGCCAGACACCUGGAGGA
361
1731
UGGCCAGACACCUGGAGGA
361
1753
UCCUCCAGGUGUCUGGCCA
561

1749
AAGUAGAACCCGAGAUGCC
362
1749
AAGUAGAACCCGAGAUGCC
362
1771
GGCAUCUCGGGUUCUACUU
562

1767
CCUGUGUCCCCCAGCUCUC
363
1767
CCUGUGUCCCCCAGCUCUC
363
1789
GAGAGCUGGGGGACACAGG
563

1785
CUGAGCCAACCACUGUGCC
364
1785
CUGAGCCAACCACUGUGCC
364
1807
GGCACAGUGGUUGGCUCAG
564

1803
CCCAACCUGAGCCAGAAAC
365
1803
CCCAACCUGAGCCAGAAAC
365
1825
GUUUCUGGCUCAGGUUGGG
565

1821
CCUGGGAGCAGAUCCUCCG
366
1821
CCUGGGAGCAGAUCCUCCG
366
1843
CGGAGGAUCUGCUCCCAGG
566

1839
GCCGAAAUGUCCUCCAGCA
367
1839
GCCGAAAUGUCCUCCAGCA
367
1861
UGCUGGAGGACAUUUCGGC
567

1857
AUGGGGCAGCUGCAGCCCC
368
1857
AUGGGGCAGCUGCAGCCCC
368
1879
GGGGCUGCAGCUGCCCCAU
568

1875
CCGUCUCGGCCCCCACCAG
369
1875
CCGUCUCGGCCCCCACCAG
369
1897
CUGGUGGGGGCCGAGACGG
569

1893
GUGGCUAUCAGGAGUUUGU
370
1893
GUGGCUAUCAGGAGUUUGU
370
1915
ACAAACUCCUGAUAGCCAC
570

1911
UACAUGCGGUGGAGCAGGG
371
1911
UACAUGCGGUGGAGCAGGG
371
1933
CCCUGCUCCACCGCAUGUA
571

1929
GUGGCACCCAGGCCAGUGC
372
1929
GUGGCACCCAGGCCAGUGC
372
1951
GCACUGGCCUGGGUGCCAC
572

1947
CGGUGGUGGGCUUGGGUCC
373
1947
CGGUGGUGGGCUUGGGUCC
373
1969
GGACCCAAGCCCACCACCG
573

1965
CCCCAGGAGAGGCUGGUUA
374
1965
CCCCAGGAGAGGCUGGUUA
374
1987
UAACCAGCCUCUCCUGGGG
574

1983
ACAAGGCCUUCUCAAGCCU
375
1983
ACAAGGCCUUCUCAAGCCU
375
2005
AGGCUUGAGAAGGCCUUGU
575

2001
UGCUUGCCAGCAGUGCUGU
376
2001
UGCUUGCCAGCAGUGCUGU
376
2023
ACAGCACUGCUGGCAAGCA
576

2019
UGUCCCCAGAGAAAUGUGG
377
2019
UGUCCCCAGAGAAAUGUGG
377
2041
CCACAUUUCUCUGGGGACA
577

2037
GGUUUGGGGCUAGCAGUGG
378
2037
GGUUUGGGGCUAGCAGUGG
378
2059
CCACUGCUAGCCCCAAACC
578

2055
GGGAAGAGGGGUAUAAGCC
379
2055
GGGAAGAGGGGUAUAAGCC
379
2077
GGCUUAUACCCCUCUUCCC
579

2073
CUUUCCAAGACCUCAUUCC
380
2073
CUUUCCAAGACCUCAUUCC
380
2095
GGAAUGAGGUCUUGGAAAG
580

2091
CUGGCUGCCCUGGGGACCC
381
2091
CUGGCUGCCCUGGGGACCC
381
2113
GGGUCCCCAGGGCAGCCAG
581

2109
CUGCCCCAGUCCCUGUCCC
382
2109
CUGCCCCAGUCCCUGUCCC
382
2131
GGGACAGGGACUGGGGCAG
582

2127
CCUUGUUCACCUUUGGACU
383
2127
CCUUGUUCACCUUUGGACU
383
2149
AGUCCAAAGGUGAACAAGG
583

2145
UGGACAGGGAGCCACCUCG
384
2145
UGGACAGGGAGCCACCUCG
384
2167
CGAGGUGGCUCCCUGUCCA
584

2163
GCAGUCCGCAGAGCUCACA
385
2163
GCAGUCCGCAGAGCUCACA
385
2185
UGUGAGCUCUGCGGACUGC
585

2181
AUCUCCCAAGCAGCUCCCC
386
2181
AUCUCCCAAGCAGCUCCCC
386
2203
GGGGAGCUGCUUGGGAGAU
586

2199
CAGAGCACCUGGGUCUGGA
387
2199
CAGAGCACCUGGGUCUGGA
387
2221
UCCAGACCCAGGUGCUCUG
587

2217
AGCCGGGGGAAAAGGUAGA
388
2217
AGCCGGGGGAAAAGGUAGA
388
2239
UCUACCUUUUCCCCCGGCU
588

2235
AGGACAUGCCAAAGCCCCC
389
2235
AGGACAUGCCAAAGCCCCC
389
2257
GGGGGCUUUGGCAUGUCCU
589

2253
CACUUCCCCAGGAGCAGGC
390
2253
CACUUCCCCAGGAGCAGGC
390
2275
GCCUGCUCCUGGGGAAGUG
590

2271
CCACAGACCCCCUUGUGGA
391
2271
CCACAGACCCCCUUGUGGA
391
2293
UCCACAAGGGGGUCUGUGG
591

2289
ACAGCCUGGGCAGUGGCAU
392
2289
ACAGCCUGGGCAGUGGCAU
392
2311
AUGCCACUGCCCAGGCUGU
592

2307
UUGUCUACUCAGCCCUUAC
393
2307
UUGUCUACUCAGCCCUUAC
393
2329
GUAAGGGCUGAGUAGACAA
593

2325
CCUGCCACCUGUGCGGCCA
394
2325
CCUGCCACCUGUGCGGCCA
394
2347
UGGCCGCACAGGUGGCAGG
594

2343
ACCUGAAACAGUGUCAUGG
395
2343
ACCUGAAACAGUGUCAUGG
395
2365
CCAUGACACUGUUUCAGGU
595

2361
GCCAGGAGGAUGGUGGCCA
396
2361
GCCAGGAGGAUGGUGGCCA
396
2383
UGGCCACCAUCCUCCUGGC
596

2379
AGACCCCUGUCAUGGCCAG
397
2379
AGACCCCUGUCAUGGCCAG
397
2401
CUGGCCAUGACAGGGGUCU
597

2397
GUCCUUGCUGUGGCUGCUG
398
2397
GUCCUUGCUGUGGCUGCUG
398
2419
CAGCAGCCACAGCAAGGAC
598

2415
GCUGUGGAGACAGGUCCUC
399
2415
GCUGUGGAGACAGGUCCUC
399
2437
GAGGACCUGUCUCCACAGC
599

2433
CGCCCCCUACAACCCCCCU
400
2433
CGCCCCCUACAACCCCCCU
400
2455
AGGGGGGUUGUAGGGGGCG
600

2451
UGAGGGCCCCAGACCCCUC
401
2451
UGAGGGCCCCAGACCCCUC
401
2473
GAGGGGUCUGGGGCCCUCA
601

2469
CUCCAGGUGGGGUUCCACU
402
2469
CUCCAGGUGGGGUUCCACU
402
2491
AGUGGAACCCCACCUGGAG
602

2487
UGGAGGCCAGUCUGUGUCC
403
2487
UGGAGGCCAGUCUGUGUCC
403
2509
GGACACAGACUGGCCUCCA
603

2505
CGGCCUCCCUGGCACCCUC
404
2505
CGGCCUCCCUGGCACCCUC
404
2527
GAGGGUGCCAGGGAGGCCG
604

2523
CGGGCAUCUCAGAGAAGAG
405
2523
CGGGCAUCUCAGAGAAGAG
405
2545
CUCUUCUCUGAGAUGCCCG
605

2541
GUAAAUCCUCAUCAUCCUU
406
2541
GUAAAUCCUCAUCAUCCUU
406
2563
AAGGAUGAUGAGGAUUUAC
606

2559
UCCAUCCUGCCCCUGGCAA
407
2559
UCCAUCCUGCCCCUGGCAA
407
2581
UUGCCAGGGGCAGGAUGGA
607

2577
AUGCUCAGAGCUCAAGCCA
408
2577
AUGCUCAGAGCUCAAGCCA
408
2599
UGGCUUGAGCUCUGAGCAU
608

2595
AGACCCCCAAAAUCGUGAA
409
2595
AGACCCCCAAAAUCGUGAA
409
2617
UUCACGAUUUUGGGGGUCU
609

2613
ACUUUGUCUCCGUGGGACC
410
2613
ACUUUGUCUCCGUGGGACC
410
2635
GGUCCCACGGAGACAAAGU
610

2631
CCACAUACAUGAGGGUCUC
411
2631
CCACAUACAUGAGGGUCUC
411
2653
GAGACCCUCAUGUAUGUGG
611

2649
CUUAGGUGCAUGUCCUCUU
412
2649
CUUAGGUGCAUGUCCUCUU
412
2671
AAGAGGACAUGCACCUAAG
612

2667
UGUUGCUGAGUCUGCAGAU
413
2667
UGUUGCUGAGUCUGCAGAU
413
2689
AUCUGCAGACUCAGCAACA
613

2685
UGAGGACUAGGGCUUAUCC
414
2685
UGAGGACUAGGGCUUAUCC
414
2707
GGAUAAGCCCUAGUCCUCA
614

2703
CAUGCCUGGGAAAUGCCAC
415
2703
CAUGCCUGGGAAAUGCCAC
415
2725
GUGGCAUUUCCCAGGCAUG
615

2721
CCUCCUGGAAGGCAGCCAG
416
2721
CCUCCUGGAAGGCAGCCAG
416
2743
CUGGCUGCCUUCCAGGAGG
616

2739
GGCUGGCAGAUUUCCAAAA
417
2739
GGCUGGCAGAUUUCCAAAA
417
2761
UUUUGGAAAUCUGCCAGCC
617

2757
AGACUUGAAGAACCAUGGU
418
2757
AGACUUGAAGAACCAUGGU
418
2779
ACCAUGGUUCUUCAAGUCU
618

2775
UAUGAAGGUGAUUGGCCCC
419
2775
UAUGAAGGUGAUUGGCCCC
419
2797
GGGGCCAAUCACCUUCAUA
619

2793
CACUGACGUUGGCCUAACA
420
2793
CACUGACGUUGGCCUAACA
420
2815
UGUUAGGCCAACGUCAGUG
620

2811
ACUGGGCUGCAGAGACUGG
421
2811
ACUGGGCUGCAGAGACUGG
421
2833
CCAGUCUCUGCAGCCCAGU
621

2829
GACCCCGCCCAGCAUUGGG
422
2829
GACCCCGCCCAGCAUUGGG
422
2851
CCCAAUGCUGGGCGGGGUC
622

2847
GCUGGGCUCGCCACAUCCC
423
2847
GCUGGGCUCGCCACAUCCC
423
2869
GGGAUGUGGCGAGCCCAGC
623

2865
CAUGAGAGUAGAGGGCACU
424
2865
CAUGAGAGUAGAGGGCACU
424
2887
AGUGCCCUCUACUCUCAUG
624

2883
UGGGUCGCCGUGCCCCACG
425
2883
UGGGUCGCCGUGCCCCACG
425
2905
CGUGGGGCACGGCGACCCA
625

2901
GGCAGGCCCCUGCAGGAAA
426
2901
GGCAGGCCCCUGCAGGAAA
426
2923
UUUCCUGCAGGGGCCUGCC
626

2919
AACUGAGGCCCUUGGGCAC
427
2919
AACUGAGGCCCUUGGGCAC
427
2941
GUGCCCAAGGGCCUCAGUU
627

2937
CCUCGACUUGUGAACGAGU
428
2937
CCUCGACUUGUGAACGAGU
428
2959
ACUCGUUCACAAGUCGAGG
628

2955
UUGUUGGCUGCUCCCUCCA
429
2955
UUGUUGGCUGCUCCCUCCA
429
2977
UGGAGGGAGCAGCCAACAA
629

2973
ACAGCUUCUGCAGCAGACU
430
2973
ACAGCUUCUGCAGCAGACU
430
2995
AGUCUGCUGCAGAAGCUGU
630

2991
UGUCCCUGUUGUAACUGCC
431
2991
UGUCCCUGUUGUAACUGCC
431
3013
GGCAGUUACAACAGGGACA
631

3009
CCAAGGCAUGUUUUGCCCA
432
3009
CCAAGGCAUGUUUUGCCCA
432
3031
UGGGCAAAACAUGCCUUGG
632

3027
ACCAGAUCAUGGCCCACGU
433
3027
ACCAGAUCAUGGCCCACGU
433
3049
ACGUGGGCCAUGAUCUGGU
633

3045
UGGAGGCCCACCUGCCUCU
434
3045
UGGAGGCCCACCUGCCUCU
434
3067
AGAGGCAGGUGGGCCUCCA
634

3063
UGUCUCACUGAACUAGAAG
435
3063
UGUCUCACUGAACUAGAAG
435
3085
CUUCUAGUUCAGUGAGACA
635

3081
GCCGAGCCUAGAAACUAAC
436
3081
GCCGAGCCUAGAAACUAAC
436
3103
GUUAGUUUCUAGGCUCGGC
636

3099
CACAGCCAUCAAGGGAAUG
437
3099
CACAGCCAUCAAGGGAAUG
437
3121
CAUUCCCUUGAUGGCUGUG
637

3117
GACUUGGGCGGCCUUGGGA
438
3117
GACUUGGGCGGCCUUGGGA
438
3139
UCCCAAGGCCGCCCAAGUC
638

3135
AAAUCGAUGAGAAAUUGAA
439
3135
AAAUCGAUGAGAAAUUGAA
439
3157
UUCAAUUUCUCAUCGAUUU
639

3153
ACUUCAGGGAGGGUGGUCA
440
3153
ACUUCAGGGAGGGUGGUCA
440
3175
UGACCACCCUCCCUGAAGU
640

3171
AUUGCCUAGAGGUGCUCAU
441
3171
AUUGCCUAGAGGUGCUCAU
441
3193
AUGAGCACCUCUAGGCAAU
641

3189
UUCAUUUAACAGAGCUUCC
442
3189
UUCAUUUAACAGAGCUUCC
442
3211
GGAAGCUCUGUUAAAUGAA
642

3207
CUUAGGUUGAUGCUGGAGG
443
3207
CUUAGGUUGAUGCUGGAGG
443
3229
CCUCCAGCAUCAACCUAAG
643

3225
GCAGAAUCCCGGCUGUCAA
444
3225
GCAGAAUCCCGGCUGUCAA
444
3247
UUGACAGCCGGGAUUCUGC
644

3243
AGGGGUGUUCAGUUAAGGG
445
3243
AGGGGUGUUCAGUUAAGGG
445
3265
CCCUUAACUGAACACCCCU
645

3261
GGAGCAACAGAGGACAUGA
446
3261
GGAGCAACAGAGGACAUGA
446
3283
UCAUGUCCUCUGUUGCUCC
646

3279
AAAAAUUGCUAUGACUAAA
447
3279
AAAAAUUGCUAUGACUAAA
447
3301
UUUAGUCAUAGCAAUUUUU
647

3297
AGCAGGGACAAUUUGCUGC
448
3297
AGCAGGGACAAUUUGCUGC
448
3319
GCAGCAAAUUGUCCCUGCU
648

3315
CCAAACACCCAUGCCCAGC
449
3315
CCAAACACCCAUGCCCAGC
449
3337
GCUGGGCAUGGGUGUUUGG
649

3333
CUGUAUGGCUGGGGGCUCC
450
3333
CUGUAUGGCUGGGGGCUCC
450
3355
GGAGCCCCCAGCCAUACAG
650

3351
CUCGUAUGCAUGGAACCCC
451
3351
CUCGUAUGCAUGGAACCCC
451
3373
GGGGUUCCAUGCAUACGAG
651

3369
CCAGAAUAAAUAUGCUCAG
452
3369
CCAGAAUAAAUAUGCUCAG
452
3391
CUGAGCAUAUUUAUUCUGG
652

3387
GCCACCCUGUGGGCCGGGC
453
3387
GCCACCCUGUGGGCCGGGC
453
3409
GCCCGGCCCACAGGGUGGC
653

3405
CAAUCCAGACAGCAGGCAU
454
3405
CAAUCCAGACAGCAGGCAU
454
3427
AUGCCUGCUGUCUGGAUUG
654

3423
UAAGGCACCAGUUACCCUG
455
3423
UAAGGCACCAGUUACCCUG
455
3445
CAGGGUAACUGGUGCCUUA
655

3441
GCAUGUUGGCCCAGACCUC
456
3441
GCAUGUUGGCCCAGACCUC
456
3463
GAGGUCUGGGCCAACAUGC
656

3459
CAGGUGCUAGGGAAGGCGG
457
3459
CAGGUGCUAGGGAAGGCGG
457
3481
CCGCCUUCCCUAGCACCUG
657

3477
GGAACCUUGGGUUGAGUAA
458
3477
GGAACCUUGGGUUGAGUAA
458
3499
UUACUCAACCCAAGGUUCC
658

3495
AUGCUCGUCUGUGUGUUUU
459
3495
AUGCUCGUCUGUGUGUUUU
459
3517
AAAACACACAGACGAGCAU
659

3513
UAGUUUCAUCACCUGUUAU
460
3513
UAGUUUCAUCACCUGUUAU
460
3535
AUAACAGGUGAUGAAACUA
660

3531
UCUGUGUUUGCUGAGGAGA
461
3531
UCUGUGUUUGCUGAGGAGA
461
3553
UCUCCUCAGCAAACACAGA
661

3549
AGUGGAACAGAAGGGGUGG
462
3549
AGUGGAACAGAAGGGGUGG
462
3571
CCACCCCUUCUGUUCCACU
662

3567
GAGUUUUGUAUAAAUAAAG
463
3567
GAGUUUUGUAUAAAUAAAG
463
3589
CUUUAUUUAUACAAAACUC
663

3577
UAAAUAAAGUUUCUUUGUC
464
3577
UAAAUAAAGUUUCUUUGUC
464
3599
GACAAAGAAACUUUAUUUA
664

IL13 NM_002188

3
GCCACCCAGCCUAUGCAUC
665
3
GCCACCCAGCCUAUGCAUC
665
25
GAUGCAUAGGCUGGGUGGC
736

21
CCGCUCCUCAAUCCUCUCC
666
21
CCGCUCCUCAAUCCUCUCC
666
43
GGAGAGGAUUGAGGAGCGG
737

39
CUGUUGGCACUGGGCCUCA
667
39
CUGUUGGCACUGGGCCUCA
667
61
UGAGGCCCAGUGCCAACAG
738

57
AUGGCGCUUUUGUUGACCA
668
57
AUGGCGCUUUUGUUGACCA
668
79
UGGUCAACAAAAGCGCCAU
739

75
ACGGUCAUUGCUCUCACUU
669
75
ACGGUCAUUGCUCUCACUU
669
97
AAGUGAGAGCAAUGACCGU
740

93
UGCCUUGGCGGCUUUGCCU
670
93
UGCCUUGGCGGCUUUGCCU
670
115
AGGCAAAGCCGCCAAGGCA
741

111
UCCCCAGGCCCUGUGCCUC
671
111
UCCCCAGGCCCUGUGCCUC
671
133
GAGGCACAGGGCCUGGGGA
742

129
CCCUCUACAGCCCUCAGGG
672
129
CCCUCUACAGCCCUCAGGG
672
151
CCCUGAGGGCUGUAGAGGG
743

147
GAGCUCAUUGAGGAGCUGG
673
147
GAGCUCAUUGAGGAGCUGG
673
169
CCAGCUCCUCAAUGAGCUC
744

165
GUCAACAUCACCCAGAACC
674
165
GUCAACAUCACCCAGAACC
674
187
GGUUCUGGGUGAUGUUGAC
745

183
CAGAAGGCUCCGCUCUGCA
675
183
CAGAAGGCUCCGCUCUGCA
675
205
UGCAGAGCGGAGCCUUCUG
746

201
AAUGGCAGCAUGGUAUGGA
676
201
AAUGGCAGCAUGGUAUGGA
676
223
UCCAUACCAUGCUGCCAUU
747

219
AGCAUCAACCUGACAGCUG
677
219
AGCAUCAACCUGACAGCUG
677
241
CAGCUGUCAGGUUGAUGCU
748

237
GGCAUGUACUGUGCAGCCC
678
237
GGCAUGUACUGUGCAGCCC
678
259
GGGCUGCACAGUACAUGCC
749

255
CUGGAAUCCCUGAUCAACG
679
255
CUGGAAUCCCUGAUCAACG
679
277
CGUUGAUCAGGGAUUCCAG
750

273
GUGUCAGGCUGCAGUGCCA
680
273
GUGUCAGGCUGCAGUGCCA
680
295
UGGCACUGCAGCCUGACAC
751

291
AUCGAGAAGACCCAGAGGA
681
291
AUCGAGAAGACCCAGAGGA
681
313
UCCUCUGGGUCUUCUCGAU
752

309
AUGCUGAGCGGAUUCUGCC
682
309
AUGCUGAGCGGAUUCUGCC
682
331
GGCAGAAUCCGCUCAGCAU
753

327
CCGCACAAGGUCUCAGCUG
683
327
CCGCACAAGGUCUCAGCUG
683
349
CAGCUGAGACCUUGUGCGG
754

345
GGGCAGUUUUCCAGCUUGC
684
345
GGGCAGUUUUCCAGCUUGC
684
367
GCAAGCUGGAAAACUGCCC
755

363
CAUGUCCGAGACACCAAAA
685
363
CAUGUCCGAGACACCAAAA
685
385
UUUUGGUGUCUCGGACAUG
756

381
AUCGAGGUGGCCCAGUUUG
686
381
AUCGAGGUGGCCCAGUUUG
686
403
CAAACUGGGCCACCUCGAU
757

399
GUAAAGGACCUGCUCUUAC
687
399
GUAAAGGACCUGCUCUUAC
687
421
GUAAGAGCAGGUCCUUUAC
758

417
CAUUUAAAGAAACUUUUUC
688
417
CAUUUAAAGAAACUUUUUC
688
439
GAAAAAGUUUCUUUAAAUG
759

435
CGCGAGGGACAGUUCAACU
689
435
CGCGAGGGACAGUUCAACU
689
457
AGUUGAACUGUCCCUCGCG
760

453
UGAAACUUCGAAAGCAUCA
690
453
UGAAACUUCGAAAGCAUCA
690
475
UGAUGCUUUCGAAGUUUCA
761

471
AUUAUUUGCAGAGACAGGA
691
471
AUUAUUUGCAGAGACAGGA
691
493
UCCUGUCUCUGCAAAUAAU
762

489
ACCUGACUAUUGAAGUUGC
692
489
ACCUGACUAUUGAAGUUGC
692
511
GCAACUUCAAUAGUCAGGU
763

507
CAGAUUCAUUUUUCUUUCU
693
507
CAGAUUCAUUUUUCUUUCU
693
529
AGAAAGAAAAAUGAAUCUG
764

525
UGAUGUCAAAAAUGUCUUG
694
525
UGAUGUCAAAAAUGUCUUG
694
547
CAAGACAUUUUUGACAUCA
765

543
GGGUAGGCGGGAAGGAGGG
695
543
GGGUAGGCGGGAAGGAGGG
695
565
CCCUCCUUCCCGCCUACCC
766

561
GUUAGGGAGGGGUAAAAUU
696
561
GUUAGGGAGGGGUAAAAUU
696
583
AAUUUUACCCCUCCCUAAC
767

579
UCCUUAGCUUAGACCUCAG
697
579
UCCUUAGCUUAGACCUCAG
697
601
CUGAGGUCUAAGCUAAGGA
768

597
GCCUGUGCUGCCCGUCUUC
698
597
GCCUGUGCUGCCCGUCUUC
698
619
GAAGACGGGCAGCACAGGC
769

615
CAGCCUAGCCGACCUCAGC
699
615
CAGCCUAGCCGACCUCAGC
699
637
GCUGAGGUCGGCUAGGCUG
770

633
CCUUCCCCUUGCCCAGGGC
700
633
CCUUCCCCUUGCCCAGGGC
700
655
GCCCUGGGCAAGGGGAAGG
771

651
CUCAGCCUGGUGGGCCUCC
701
651
CUCAGCCUGGUGGGCCUCC
701
673
GGAGGCCCACCAGGCUGAG
772

669
CUCUGUCCAGGGCCCUGAG
702
669
CUCUGUCCAGGGCCCUGAG
702
691
CUCAGGGCCCUGGACAGAG
773

687
GCUCGGUGGACCCAGGGAU
703
687
GCUCGGUGGACCCAGGGAU
703
709
AUCCCUGGGUCCACCGAGC
774

705
UGACAUGUCCCUACACCCC
704
705
UGACAUGUCCCUACACCCC
704
727
GGGGUGUAGGGACAUGUCA
775

723
CUCCCCUGCCCUAGAGCAC
705
723
CUCCCCUGCCCUAGAGCAC
705
745
GUGCUCUAGGGCAGGGGAG
776

741
CACUGUAGCAUUACAGUGG
706
741
CACUGUAGCAUUACAGUGG
706
763
CCACUGUAAUGCUACAGUG
777

759
GGUGCCCCCCUUGCCAGAC
707
759
GGUGCCCCCCUUGCCAGAC
707
781
GUCUGGCAAGGGGGGCACC
778

777
CAUGUGGUGGGACAGGGAC
708
777
CAUGUGGUGGGACAGGGAC
708
799
GUCCCUGUCCCACCACAUG
779

795
CCCACUUCACACACAGGCA
709
795
CCCACUUCACACACAGGCA
709
817
UGCCUGUGUGUGAAGUGGG
780

813
AACUGAGGCAGACAGCAGC
710
813
AACUGAGGCAGACAGCAGC
710
835
GCUGCUGUCUGCCUCAGUU
781

831
CUCAGGCACACUUCUUCUU
711
831
CUCAGGCACACUUCUUCUU
711
853
AAGAAGAAGUGUGCCUGAG
782

849
UGGUCUUAUUUAUUAUUGU
712
849
UGGUCUUAUUUAUUAUUGU
712
871
ACAAUAAUAAAUAAGACCA
783

867
UGUGUUAUUUAAAUGAGUG
713
867
UGUGUUAUUUAAAUGAGUG
713
889
CACUCAUUUAAAUAACACA
784

885
GUGUUUGUCACCGUUGGGG
714
885
GUGUUUGUCACCGUUGGGG
714
907
CCCCAACGGUGACAAACAC
785

903
GAUUGGGGAAGACUGUGGC
715
903
GAUUGGGGAAGACUGUGGC
715
925
GCCACAGUCUUCCCCAAUC
786

921
CUGCUAGCACUUGGAGCCA
716
921
CUGCUAGCACUUGGAGCCA
716
943
UGGCUCCAAGUGCUAGCAG
787

939
AAGGGUUCAGAGACUCAGG
717
939
AAGGGUUCAGAGACUCAGG
717
961
CCUGAGUCUCUGAACCCUU
788

957
GGCCCCAGCACUAAAGCAG
718
957
GGCCCCAGCACUAAAGCAG
718
979
CUGCUUUAGUGCUGGGGCC
789

975
GUGGACACCAGGAGUCCCU
719
975
GUGGACACCAGGAGUCCCU
719
997
AGGGACUCCUGGUGUCCAC
790

993
UGGUAAUAAGUACUGUGUA
720
993
UGGUAAUAAGUACUGUGUA
720
1015
UACACAGUACUUAUUACCA
791

1011
ACAGAAUUCUGCUACCUCA
721
1011
ACAGAAUUCUGCUACCUCA
721
1033
UGAGGUAGCAGAAUUCUGU
792

1029
ACUGGGGUCCUGGGGCCUC
722
1029
ACUGGGGUCCUGGGGCCUC
722
1051
GAGGCCCCAGGACCCCAGU
793

1047
CGGAGCCUCAUCCGAGGCA
723
1047
CGGAGCCUCAUCCGAGGCA
723
1069
UGCCUCGGAUGAGGCUCCG
794

1055
AGGGUCAGGAGAGGGGCAG
724
1055
AGGGUCAGGAGAGGGGCAG
724
1087
CUGCCCCUCUCCUGACCCU
795

1083
GAACAGCCGCUCCUGUCUG
725
1083
GAACAGCCGCUCCUGUCUG
725
1105
CAGACAGGAGCGGCUGUUC
796

1101
GCCAGCCAGCAGCCAGCUC
726
1101
GCCAGCCAGCAGCCAGCUC
726
1123
GAGCUGGCUGCUGGCUGGC
797

1119
CUCAGCCAACGAGUAAUUU
727
1119
CUCAGCCAACGAGUAAUUU
727
1141
AAAUUACUCGUUGGCUGAG
798

1137
UAUUGUUUUUCCUUGUAUU
728
1137
UAUUGUUUUUCCUUGUAUU
728
1159
AAUACAAGGAAAAACAAUA
799

1155
UUAAAUAUUAAAUAUGUUA
729
1155
UUAAAUAUUAAAUAUGUUA
729
1177
UAACAUAUUUAAUAUUUAA
800

1173
AGCAAAGAGUUAAUAUAUA
730
1173
AGCAAAGAGUUAAUAUAUA
730
1195
UAUAUAUUAACUCUUUGCU
801

1191
AGAAGGGUACCUUGAACAC
731
1191
AGAAGGGUACCUUGAACAC
731
1213
GUGUUCAAGGUACCCUUCU
802

1209
CUGGGGGAGGGGACAUUGA
732
1209
CUGGGGGAGGGGACAUUGA
732
1231
UCAAUGUCCCCUCCCCCAG
803

1227
AACAAGUUGUUUCAUUGAC
733
1227
AACAAGUUGUUUCAUUGAC
733
1249
GUCAAUGAAACAACUUGUU
804

1245
CUAUCAAACUGAAGCCAGA
734
1245
CUAUCAAACUGAAGCCAGA
734
1267
UCUGGCUUCAGUUUGAUAG
805

1262
GAAAUAAAGUUGGUGACAG
735
1262
GAAAUAAAGUUGGUGACAG
735
1284
CUGUCACCAACUUUAUUUC
806

IL13RA1 NM_001560

3
CCAAGGCUCCAGCCCGGCC
807
3
CCAAGGCUCCAGCCCGGCC
807
25
GGCCGGGCUGGAGCCUUGG
1030

21
CGGGCUCCGAGGCGAGAGG
808
21
CGGGCUCCGAGGCGAGAGG
808
43
CCUCUCGCCUCGGAGCCCG
1031

39
GCUGCAUGGAGUGGCCGGC
809
39
GCUGCAUGGAGUGGCCGGC
809
61
GCCGGGCACUCCAUGCAGC
1032

57
CGCGGCUCUGCGGGCUGUG
810
57
CGCGGCUCUGCGGGCUGUG
810
79
CACAGCCCGCAGAGCCGCG
1033

75
GGGCGCUGCUGCUCUGCGC
811
75
GGGCGCUGCUGCUCUGCGC
811
97
GCGCAGAGCAGCAGCGCCC
1034

93
CCGGCGGCGGGGGCGGGGG
812
93
CCGGCGGCGGGGGCGGGGG
812
115
CCCCCGCCCCCGCCGCCGG
1035

111
GCGGGGGCGCCGCGCCUAC
813
111
GCGGGGGCGCCGCGCCUAC
813
133
GUAGGCGCGGCGCCCCCGC
1036

129
CGGAAACUCAGCCACCUGU
814
129
CGGAAACUCAGCCACCUGU
814
151
ACAGGUGGCUGAGUUUCCG
1037

147
UGACAAAUUUGAGUGUCUC
815
147
UGACAAAUUUGAGUGUCUC
815
169
GAGACACUCAAAUUUGUCA
1038

165
CUGUUGAAAACCUCUGCAC
816
165
CUGUUGAAAACCUCUGCAC
816
187
GUGCAGAGGUUUUCAACAG
1039

183
CAGUAAUAUGGACAUGGAA
817
183
CAGUAAUAUGGACAUGGAA
817
205
UUCCAUGUCCAUAUUACUG
1040

201
AUCCACCCGAGGGAGCCAG
818
201
AUCCACCCGAGGGAGCCAG
818
223
CUGGCUCCCUCGGGUGGAU
1041

219
GCUCAAAUUGUAGUCUAUG
819
219
GCUCAAAUUGUAGUCUAUG
819
241
CAUAGACUACAAUUUGAGC
1042

237
GGUAUUUUAGUCAUUUUGG
820
237
GGUAUUUUAGUCAUUUUGG
820
259
CCAAAAUGACUAAAAUACC
1043

255
GCGACAAACAAGAUAAGAA
821
255
GCGACAAACAAGAUAAGAA
821
277
UUCUUAUCUUGUUUGUCGC
1044

273
AAAUAGCUCCGGAAACUCG
822
273
AAAUAGCUCCGGAAACUCG
822
295
CGAGUUUCCGGAGCUAUUU
1045

291
GUCGUUCAAUAGAAGUACC
823
291
GUCGUUCAAUAGAAGUACC
823
313
GGUACUUCUAUUGAACGAC
1046

309
CCCUGAAUGAGAGGAUUUG
824
309
CCCUGAAUGAGAGGAUUUG
824
331
CAAAUCCUCUCAUUCAGGG
1047

327
GUCUGCAAGUGGGGUCCCA
825
327
GUCUGCAAGUGGGGUCCCA
825
349
UGGGACCCCACUUGCAGAC
1048

345
AGUGUAGCACCAAUGAGAG
826
345
AGUGUAGCACCAAUGAGAG
826
367
CUCUCAUUGGUGCUACACU
1049

363
GUGAGAAGCCUAGCAUUUU
827
363
GUGAGAAGCCUAGCAUUUU
827
385
AAAAUGCUAGGCUUCUCAC
1050

381
UGGUUGAAAAAUGCAUCUC
828
381
UGGUUGAAAAAUGCAUCUC
828
403
GAGAUGCAUUUUUCAACCA
1051

399
CACCCCCAGAAGGUGAUCC
829
399
CACCCCCAGAAGGUGAUCC
829
421
GGAUCACCUUCUGGGGGUG
1052

417
CUGAGUCUGCUGUGACUGA
830
417
CUGAGUCUGCUGUGACUGA
830
439
UCAGUCACAGCAGACUCAG
1053

435
AGCUUCAAUGCAUUUGGCA
831
435
AGCUUCAAUGCAUUUGGCA
831
457
UGCCAAAUGCAUUGAAGCU
1054

453
ACAACCUGAGCUACAUGAA
832
453
ACAACCUGAGCUACAUGAA
832
475
UUCAUGUAGCUCAGGUUGU
1055

471
AGUGUUCUUGGCUCCCUGG
833
471
AGUGUUCUUGGCUCCCUGG
833
493
CCAGGGAGCCAAGAACACU
1056

489
GAAGGAAUACCAGUCCCGA
834
489
GAAGGAAUACCAGUCCCGA
834
511
UCGGGACUGGUAUUCCUUC
1057

507
ACACUAACUAUACUCUCUA
835
507
ACACUAACUAUACUCUCUA
835
529
UAGAGAGUAUAGUUAGUGU
1058

525
ACUAUUGGCACAGAAGCCU
836
525
ACUAUUGGCACAGAAGCCU
836
547
AGGCUUCUGUGCCAAUAGU
1059

543
UGGAAAAAAUUCAUCAAUG
837
543
UGGAAAAAAUUCAUCAAUG
837
565
CAUUGAUGAAUUUUUUCCA
1060

561
GUGAAAACAUCUUUAGAGA
838
561
GUGAAAACAUCUUUAGAGA
838
583
UCUCUAAAGAUGUUUUCAC
1061

579
AAGGCCAAUACUUUGGUUG
839
579
AAGGCCAAUACUUUGGUUG
839
601
CAACCAAAGUAUUGGCCUU
1062

597
GUUCCUUUGAUCUGACCAA
840
597
GUUCCUUUGAUCUGACCAA
840
619
UUGGUCAGAUCAAAGGAAC
1063

615
AAGUGAAGGAUUCCAGUUU
841
615
AAGUGAAGGAUUCCAGUUU
841
637
AAACUGGAAUCCUUCACUU
1064

633
UUGAACAACACAGUGUCCA
842
633
UUGAACAACACAGUGUCCA
842
655
UGGACACUGUGUUGUUCAA
1065

651
AAAUAAUGGUCAAGGAUAA
843
651
AAAUAAUGGUCAAGGAUAA
843
673
UUAUCCUUGACCAUUAUUU
1066

669
AUGCAGGAAAAAUUAAACC
844
669
AUGCAGGAAAAAUUAAACC
844
691
GGUUUAAUUUUUCCUGCAU
1067

687
CAUCCUUCAAUAUAGUGCC
845
687
CAUCCUUCAAUAUAGUGCC
845
709
GGCACUAUAUUGAAGGAUG
1068

705
CUUUAACUUCCCGUGUGAA
846
705
CUUUAACUUCCCGUGUGAA
846
727
UUCACACGGGAAGUUAAAG
1069

723
AACCUGAUCCUCCACAUAU
847
723
AACCUGAUCCUCCACAUAU
847
745
AUAUGUGGAGGAUCAGGUU
1070

741
UUAAAAACCUCUCCUUCCA
848
741
UUAAAAACCUCUCCUUCCA
848
763
UGGAAGGAGAGGUUUUUAA
1071

759
ACAAUGAUGACCUAUAUGU
849
759
ACAAUGAUGACCUAUAUGU
849
781
ACAUAUAGGUCAUCAUUGU
1072

777
UGCAAUGGGAGAAUCCACA
850
777
UGCAAUGGGAGAAUCCACA
850
799
UGUGGAUUCUCCCAUUGCA
1073

795
AGAAUUUUAUUAGCAGAUG
851
795
AGAAUUUUAUUAGCAGAUG
851
817
CAUCUGCUAAUAAAAUUCU
1074

813
GCCUAUUUUAUGAAGUAGA
852
813
GCCUAUUUUAUGAAGUAGA
852
835
UCUACUUCAUAAAAUAGGC
1075

831
AAGUCAAUAACAGCCAAAC
853
831
AAGUCAAUAACAGCCAAAC
853
853
GUUUGGCUGUUAUUGACUU
1076

849
CUGAGACACAUAAUGUUUU
854
849
CUGAGACACAUAAUGUUUU
854
871
AAAACAUUAUGUGUCUCAG
1077

867
UCUACGUCCAAGAGGCUAA
855
867
UCUACGUCCAAGAGGCUAA
855
889
UUAGCCUCUUGGACGUAGA
1078

885
AAUGUGAGAAUCCAGAAUU
856
885
AAUGUGAGAAUCCAGAAUU
856
907
AAUUCUGGAUUCUCACAUU
1079

903
UUGAGAGAAAUGUGGAGAA
857
903
UUGAGAGAAAUGUGGAGAA
857
925
UUCUCCACAUUUCUCUCAA
1080

921
AUACAUCUUGUUUCAUGGU
858
921
AUACAUCUUGUUUCAUGGU
858
943
ACCAUGAAACAAGAUGUAU
1081

939
UCCCUGGUGUUCUUCCUGA
859
939
UCCCUGGUGUUCUUCCUGA
859
961
UCAGGAAGAACACCAGGGA
1082

957
AUACUUUGAACACAGUCAG
860
957
AUACUUUGAACACAGUCAG
860
979
CUGACUGUGUUCAAAGUAU
1083

975
GAAUAAGAGUCAAAACAAA
861
975
GAAUAAGAGUCAAAACAAA
861
997
UUUGUUUUGACUCUUAUUC
1084

993
AUAAGUUAUGCUAUGAGGA
862
993
AUAAGUUAUGCUAUGAGGA
862
1015
UCCUCAUAGCAUAACUUAU
1085

1011
AUGACAAACUCUGGAGUAA
863
1011
AUGACAAACUCUGGAGUAA
863
1033
UUACUCCAGAGUUUGUCAU
1086

1029
AUUGGAGCCAAGAAAUGAG
864
1029
AUUGGAGCCAAGAAAUGAG
864
1051
CUCAUUUCUUGGCUCCAAU
1087

1047
GUAUAGGUAAGAAGCGCAA
865
1047
GUAUAGGUAAGAAGCGCAA
865
1069
UUGCGCUUCUUACCUAUAC
1088

1065
AUUCCACACUCUACAUAAC
866
1065
AUUCCACACUCUACAUAAC
866
1087
GUUAUGUAGAGUGUGGAAU
1089

1083
CCAUGUUACUCAUUGUUCC
867
1083
CCAUGUUACUCAUUGUUCC
867
1105
GGAACAAUGAGUAACAUGG
1090

1101
CAGUCAUCGUCGCAGGUGC
868
1101
CAGUCAUCGUCGCAGGUGC
868
1123
GCACCUGCGACGAUGACUG
1091

1119
CAAUCAUAGUACUCCUGCU
869
1119
CAAUCAUAGUACUCCUGCU
869
1141
AGCAGGAGUACUAUGAUUG
1092

1137
UUUACCUAAAAAGGCUCAA
870
1137
UUUACCUAAAAAGGCUCAA
870
1159
UUGAGCCUUUUUAGGUAAA
1093

1155
AGAUUAUUAUAUUCCCUCC
871
1155
AGAUUAUUAUAUUCCCUCC
871
1177
GGAGGGAAUAUAAUAAUCU
1094

1173
CAAUUCCUGAUCCUGGCAA
872
1173
CAAUUCCUGAUCCUGGCAA
872
1195
UUGCCAGGAUCAGGAAUUG
1095

1191
AGAUUUUUAAAGAAAUGUU
873
1191
AGAUUUUUAAAGAAAUGUU
873
1213
AACAUUUCUUUAAAAAUCU
1096

1209
UUGGAGACCAGAAUGAUGA
874
1209
UUGGAGACCAGAAUGAUGA
874
1231
UCAUCAUUCUGGUCUCCAA
1097

1227
AUACUCUGCACUGGAAGAA
875
1227
AUACUCUGCACUGGAAGAA
875
1249
UUCUUCCAGUGCAGAGUAU
1098

1245
AGUACGACAUCUAUGAGAA
876
1245
AGUACGACAUCUAUGAGAA
876
1267
UUCUCAUAGAUGUCGUACU
1099

1263
AGCAAACCAAGGAGGAAAC
877
1263
AGCAAACCAAGGAGGAAAC
877
1285
GUUUCCUCCUUGGUUUGCU
1100

1281
CCGACUCUGUAGUGCUGAU
878
1281
CCGACUCUGUAGUGCUGAU
878
1303
AUCAGCACUACAGAGUCGG
1101

1299
UAGAAAACCUGAAGAAAGC
879
1299
UAGAAAACCUGAAGAAAGC
879
1321
GCUUUCUUCAGGUUUUCUA
1102

1317
CCUCUCAGUGAUGGAGAUA
880
1317
CCUCUCAGUGAUGGAGAUA
880
1339
UAUCUCCAUCACUGAGAGG
1103

1335
AAUUUAUUUUUACCUUCAC
881
1335
AAUUUAUUUUUACCUUCAC
881
1357
GUGAAGGUAAAAAUAAAUU
1104

1353
CUGUGACCUUGAGAAGAUU
882
1353
CUGUGACCUUGAGAAGAUU
882
1375
AAUCUUCUCAAGGUCACAG
1105

1371
UCUUCCCAUUCUCCAUUUG
883
1371
UCUUCCCAUUCUCCAUUUG
883
1393
CAAAUGGAGAAUGGGAAGA
1106

1389
GUUAUCUGGGAACUUAUUA
884
1389
GUUAUCUGGGAACUUAUUA
884
1411
UAAUAAGUUCCCAGAUAAC
1107

1407
AAAUGGAAACUGAAACUAC
885
1407
AAAUGGAAACUGAAACUAC
885
1429
GUAGUUUCAGUUUCCAUUU
1108

1425
CUGCACCAUUUAAAAACAG
886
1425
CUGCACCAUUUAAAAACAG
886
1447
CUGUUUUUAAAUGGUGCAG
1109

1443
GGCAGCUCAUAAGAGCCAC
887
1443
GGCAGCUCAUAAGAGCCAC
887
1465
GUGGCUCUUAUGAGCUGCC
1110

1461
CAGGUCUUUAUGUUGAGUC
888
1461
CAGGUCUUUAUGUUGAGUC
888
1483
GACUCAACAUAAAGACCUG
1111

1479
CGCGCACCGAAAAACUAAA
889
1479
CGCGCACCGAAAAACUAAA
889
1501
UUUAGUUUUUCGGUGCGCG
1112

1497
AAAUAAUGGGCGCUUUGGA
890
1497
AAAUAAUGGGCGCUUUGGA
890
1519
UCCAAAGCGCCCAUUAUUU
1113

1515
AGAAGAGUGUGGAGUCAUU
891
1515
AGAAGAGUGUGGAGUCAUU
891
1537
AAUGACUCCACACUCUUCU
1114

1533
UCUCAUUGAAUUAUAAAAG
892
1533
UCUCAUUGAAUUAUAAAAG
892
1555
CUUUUAUAAUUCAAUGAGA
1115

1551
GCCAGCAGGCUUCAAACUA
893
1551
GCCAGCAGGCUUCAAACUA
893
1573
UAGUUUGAAGCCUGCUGGC
1116

1569
AGGGGACAAAGCAAAAAGU
894
1569
AGGGGACAAAGCAAAAAGU
894
1591
ACUUUUUGCUUUGUCCCCU
1117

1587
UGAUGAUAGUGGUGGAGUU
895
1587
UGAUGAUAGUGGUGGAGUU
895
1609
AACUCCACCACUAUCAUCA
1118

1605
UAAUCUUAUCAAGAGUUGU
896
1605
UAAUCUUAUCAAGAGUUGU
896
1627
ACAACUCUUGAUAAGAUUA
1119

1623
UGACAACUUCCUGAGGGAU
897
1623
UGACAACUUCCUGAGGGAU
897
1645
AUCCCUCAGGAAGUUGUCA
1120

1641
UCUAUACUUGCUUUGUGUU
898
1641
UCUAUACUUGCUUUGUGUU
898
1663
AACACAAAGCAAGUAUAGA
1121

1659
UCUUUGUGUCAACAUGAAC
899
1659
UCUUUGUGUCAACAUGAAC
899
1681
GUUCAUGUUGACACAAAGA
1122

1677
CAAAUUUUAUUUGUAGGGG
900
1677
CAAAUUUUAUUUGUAGGGG
900
1699
CCCCUACAAAUAAAAUUUG
1123

1695
GAACUCAUUUGGGGUGCAA
901
1695
GAACUCAUUUGGGGUGCAA
901
1717
UUGCACCCCAAAUGAGUUC
1124

1713
AAUGCUAAUGUCAAACUUG
902
1713
AAUGCUAAUGUCAAACUUG
902
1735
CAAGUUUGACAUUAGCAUU
1125

1731
GAGUCACAAAGAACAUGUA
903
1731
GAGUCACAAAGAACAUGUA
903
1753
UACAUGUUCUUUGUGACUC
1126

1749
AGAAAACAAAAUGGAUAAA
904
1749
AGAAAACAAAAUGGAUAAA
904
1771
UUUAUCCAUUUUGUUUUCU
1127

1767
AAUCUGAUAUGUAUUGUUU
905
1767
AAUCUGAUAUGUAUUGUUU
905
1789
AAACAAUACAUAUCAGAUU
1128

1785
UGGGAUCCUAUUGAACCAU
906
1785
UGGGAUCCUAUUGAACCAU
906
1807
AUGGUUCAAUAGGAUCCCA
1129

1803
UGUUUGUGGCUAUUAAAAC
907
1803
UGUUUGUGGCUAUUAAAAC
907
1825
GUUUUAAUAGCCACAAACA
1130

1821
CUCUUUUAACAGUCUGGGC
908
1821
CUCUUUUAACAGUCUGGGC
908
1843
GCCCAGACUGUUAAAAGAG
1131

1839
CUGGGUCCGGUGGCUCACG
909
1839
CUGGGUCCGGUGGCUCACG
909
1861
CGUGAGCCACCGGACCCAG
1132

1857
GCCUGUAAUCCCAGCAAUU
910
1857
GCCUGUAAUCCCAGCAAUU
910
1879
AAUUGCUGGGAUUACAGGC
1133

1875
UUGGGAGUCCGAGGCGGGC
911
1875
UUGGGAGUCCGAGGCGGGC
911
1897
GCCCGCCUCGGACUCCCAA
1134

1893
CGGAUCACUCGAGGUCAGG
912
1893
CGGAUCACUCGAGGUCAGG
912
1915
CCUGACCUCGAGUGAUCCG
1135

1911
GAGUUCCAGACCAGCCUGA
913
1911
GAGUUCCAGACCAGCCUGA
913
1933
UCAGGCUGGUCUGGAACUC
1136

1929
ACCAAAAUGGUGAAACCUC
914
1929
ACCAAAAUGGUGAAACCUC
914
1951
GAGGUUUCACCAUUUUGGU
1137

1947
CCUCUCUACUAAAACUACA
915
1947
CCUCUCUACUAAAACUACA
915
1969
UGUAGUUUUAGUAGAGAGG
1138

1965
AAAAAUUAACUGGGUGUGG
916
1965
AAAAAUUAACUGGGUGUGG
916
1987
CCACACCCAGUUAAUUUUU
1139

1983
GUGGCGCGUGCCUGUAAUC
917
1983
GUGGCGCGUGCCUGUAAUC
917
2005
GAUUACAGGCACGCGCCAC
1140

2001
CCCAGCUACUCGGGAAGCU
918
2001
CCCAGCUACUCGGGAAGCU
918
2023
AGCUUCCCGAGUAGCUGGG
1141

2019
UGAGGCAGGUGAAUUGUUU
919
2019
UGAGGCAGGUGAAUUGUUU
919
2041
AAACAAUUCACCUGCCUCA
1142

2037
UGAACCUGGGAGGUGGAGG
920
2037
UGAACCUGGGAGGUGGAGG
920
2059
CCUCCACCUCCCAGGUUCA
1143

2055
GUUGCAGUGAGCAGAGAUC
921
2055
GUUGCAGUGAGCAGAGAUC
921
2077
GAUCUCUGCUCACUGCAAC
1144

2073
CACACCACUGCACUCUAGC
922
2073
CACACCACUGCACUCUAGC
922
2095
GCUAGAGUGCAGUGGUGUG
1145

2091
CCUGGGUGACAGAGCAAGA
923
2091
CCUGGGUGACAGAGCAAGA
923
2113
UCUUGCUCUGUCACCCAGG
1146

2109
ACUCUGUCUAAAAAACAAA
924
2109
ACUCUGUCUAAAAAACAAA
924
2131
UUUGUUUUUUAGACAGAGU
1147

2127
AACAAAACAAAACAAAACA
925
2127
AACAAAACAAAACAAAACA
925
2149
UGUUUUGUUUUGUUUUGUU
1148

2145
AAAAAAACCUCUUAAUAUU
926
2145
AAAAAAACCUCUUAAUAUU
926
2167
AAUAUUAAGAGGUUUUUUU
1149

2163
UCUGGAGUCAUCAUUCCCU
927
2163
UCUGGAGUCAUCAUUCCCU
927
2185
AGGGAAUGAUGACUCCAGA
1150

2181
UUCGACAGCAUUUUCCUCU
928
2181
UUCGACAGCAUUUUCCUCU
928
2203
AGAGGAAAAUGCUGUCGAA
1151

2199
UGCUUUGAAAGCCCCAGAA
929
2199
UGCUUUGAAAGCCCCAGAA
929
2221
UUCUGGGGCUUUCAAAGCA
1152

2217
AAUCAGUGUUGGCCAUGAU
930
2217
AAUCAGUGUUGGCCAUGAU
930
2239
AUCAUGGCCAACACUGAUU
1153

2235
UGACAACUACAGAAAAACC
931
2235
UGACAACUACAGAAAAACC
931
2257
GGUUUUUCUGUAGUUGUCA
1154

2253
CAGAGGCAGCUUCUUUGCC
932
2253
CAGAGGCAGCUUCUUUGCC
932
2275
GGCAAAGAAGCUGCCUCUG
1155

2271
CAAGACCUUUCAAAGCCAU
933
2271
CAAGACCUUUCAAAGCCAU
933
2293
AUGGCUUUGAAAGGUCUUG
1156

2289
UUUUAGGCUGUUAGGGGCA
934
2289
UUUUAGGCUGUUAGGGGCA
934
2311
UGCCCCUAACAGCCUAAAA
1157

2307
AGUGGAGGUAGAAUGACUC
935
2307
AGUGGAGGUAGAAUGACUC
935
2329
GAGUCAUUCUACCUCCACU
1158

2325
CCUUGGGUAUUAGAGUUUC
936
2325
CCUUGGGUAUUAGAGUUUC
936
2347
GAAACUCUAAUACCCAAGG
1159

2343
CAACCAUGAAGUCUCUAAC
937
2343
CAACCAUGAAGUCUCUAAC
937
2365
GUUAGAGACUUCAUGGUUG
1160

2361
CAAUGUAUUUUCUUCACCU
938
2361
CAAUGUAUUUUCUUCACCU
938
2383
AGGUGAAGAAAAUACAUUG
1161

2379
UCUGCUACUCAAGUAGCAU
939
2379
UCUGCUACUCAAGUAGCAU
939
2401
AUGCUACUUGAGUAGCAGA
1162

2397
UUUACUGUGUCUUUGGUUU
940
2397
UUUACUGUGUCUUUGGUUU
940
2419
AAACCAAAGACACAGUAAA
1163

2415
UGUGCUAGGCCCCCGGGUG
941
2415
UGUGCUAGGCCCCCGGGUG
941
2437
CACCCGGGGGCCUAGCACA
1164

2433
GUGAAGCACAGACCCCUUC
942
2433
GUGAAGCACAGACCCCUUC
942
2455
GAAGGGGUCUGUGCUUCAC
1165

2451
CCAGGGGUUUACAGUCUAU
943
2451
CCAGGGGUUUACAGUCUAU
943
2473
AUAGACUGUAAACCCCUGG
1166

2469
UUUGAGACUCCUCAGUUCU
944
2469
UUUGAGACUCCUCAGUUCU
944
2491
AGAACUGAGGAGUCUCAAA
1167

2487
UUGCCACUUUUUUUUUUAA
945
2487
UUGCCACUUUUUUUUUUAA
945
2509
UUAAAAAAAAAAGUGGCAA
1168

2505
AUCUCCACCAGUCAUUUUU
946
2505
AUCUCCACCAGUCAUUUUU
946
2527
AAAAAUGACUGGUGGAGAU
1169

2523
UCAGACCUUUUAACUCCUC
947
2523
UCAGACCUUUUAACUCCUC
947
2545
GAGGAGUUAAAAGGUCUGA
1170

2541
CAAUUCCAACACUGAUUUC
948
2541
CAAUUCCAACACUGAUUUC
948
2563
GAAAUCAGUGUUGGAAUUG
1171

2559
CCCCUUUUGCAUUCUCCCU
949
2559
CCCCUUUUGCAUUCUCCCU
949
2581
AGGGAGAAUGCAAAAGGGG
1172

2577
UCCUUCCCUUCCUUGUAGC
950
2577
UCCUUCCCUUCCUUGUAGC
950
2599
GCUACAAGGAAGGGAAGGA
1173

2595
CCUUUUGACUUUCAUUGGA
951
2595
CCUUUUGACUUUCAUUGGA
951
2617
UCCAAUGAAAGUCAAAAGG
1174

2613
AAAUUAGGAUGUAAAUCUG
952
2613
AAAUUAGGAUGUAAAUCUG
952
2635
CAGAUUUACAUCCUAAUUU
1175

2631
GCUCAGGAGACCUGGAGGA
953
2631
GCUCAGGAGACCUGGAGGA
953
2653
UCCUCCAGGUCUCCUGAGC
1176

2649
AGCAGAGGAUAAUUAGCAU
954
2649
AGCAGAGGAUAAUUAGCAU
954
2671
AUGCUAAUUAUCCUCUGCU
1177

2667
UCUCAGGUUAAGUGUGAGU
955
2667
UCUCAGGUUAAGUGUGAGU
955
2689
ACUCACACUUAACCUGAGA
1178

2685
UAAUCUGAGAAACAAUGAC
956
2685
UAAUCUGAGAAACAAUGAC
956
2707
GUCAUUGUUUCUCAGAUUA
1179

2703
CUAAUUCUUGCAUAUUUUG
957
2703
CUAAUUCUUGCAUAUUUUG
957
2725
CAAAAUAUGCAAGAAUUAG
1180

2721
GUAACUUCCAUGUGAGGGU
958
2721
GUAACUUCCAUGUGAGGGU
958
2743
ACCCUCACAUGGAAGUUAC
1181

2739
UUUUCAGCAUUGAUAUUUG
959
2739
UUUUCAGCAUUGAUAUUUG
959
2761
CAAAUAUCAAUGCUGAAAA
1182

2757
GUGCAUUUUCUAAACAGAG
960
2757
GUGCAUUUUCUAAACAGAG
960
2779
CUCUGUUUAGAAAAUGCAC
1183

2775
GAUGAGGUGGUAUCUUCAC
961
2775
GAUGAGGUGGUAUCUUCAC
961
2797
GUGAAGAUACCACCUCAUC
1184

2793
CGUAGAACAUUGGUAUUCG
962
2793
CGUAGAACAUUGGUAUUCG
962
2815
CGAAUACCAAUGUUCUACG
1185

2811
GCUUGAGAAAAAAAGAAUA
963
2811
GCUUGAGAAAAAAAGAAUA
963
2833
UAUUCUUUUUUUCUCAAGC
1186

2829
AGUUGAACCUAUUUCUCUU
964
2829
AGUUGAACCUAUUUCUCUU
964
2851
AAGAGAAAUAGGUUCAACU
1187

2847
UUCUUUACAAGAUGGGUCC
965
2847
UUCUUUACAAGAUGGGUCC
965
2869
GGACCCAUCUUGUAAAGAA
1188

2865
CAGGAUUCCUCUUUUCUCU
966
2865
CAGGAUUCCUCUUUUCUCU
966
2887
AGAGAAAAGAGGAAUCCUG
1189

2883
UGCCAUAAAUGAUUAAUUA
967
2883
UGCCAUAAAUGAUUAAUUA
967
2905
UAAUUAAUCAUUUAUGGCA
1190

2901
AAAUAGCUUUUGUGUCUUA
968
2901
AAAUAGCUUUUGUGUCUUA
968
2923
UAAGACACAAAAGCUAUUU
1191

2919
ACAUUGGUAGCCAGCCAGC
969
2919
ACAUUGGUAGCCAGCCAGC
969
2941
GCUGGCUGGCUACCAAUGU
1192

2937
CCAAGGCUCUGUUUAUGCU
970
2937
CCAAGGCUCUGUUUAUGCU
970
2959
AGCAUAAACAGAGCCUUGG
1193

2955
UUUUGGGGGGCAUAUAUUG
971
2955
UUUUGGGGGGCAUAUAUUG
971
2977
CAAUAUAUGCCCCCCAAAA
1194

2973
GGGUUCCAUUCUCACCUAU
972
2973
GGGUUCCAUUCUCACCUAU
972
2995
AUAGGUGAGAAUGGAACCC
1195

2991
UCCACACAACAUAUCCGUA
973
2991
UCCACACAACAUAUCCGUA
973
3013
UACGGAUAUGUUGUGUGGA
1196

3009
AUAUAUCCCCUCUACUCUU
974
3009
AUAUAUCCCCUCUACUCUU
974
3031
AAGAGUAGAGGGGAUAUAU
1197

3027
UACUUCCCCCAAAUUUAAA
975
3027
UACUUCCCCCAAAUUUAAA
975
3049
UUUAAAUUUGGGGGAAGUA
1198

3045
AGAAGUAUGGGAAAUGAGA
976
3045
AGAAGUAUGGGAAAUGAGA
976
3067
UCUCAUUUCCCAUACUUCU
1199

3063
AGGCAUUUCCCCCACCCCA
977
3063
AGGCAUUUCCCCCACCCCA
977
3085
UGGGGUGGGGGAAAUGCCU
1200

3081
AUUUCUCUCCUCACACACA
978
3081
AUUUCUCUCCUCACACACA
978
3103
UGUGUGUGAGGAGAGAAAU
1201

3099
AGACUCAUAUUACUGGUAG
979
3099
AGACUCAUAUUACUGGUAG
979
3121
CUACCAGUAAUAUGAGUCU
1202

3117
GGAACUUGAGAACUUUAUU
980
3117
GGAACUUGAGAACUUUAUU
980
3139
AAUAAAGUUCUCAAGUUCC
1203

3135
UUCCAAGUUGUUCAAACAU
981
3135
UUCCAAGUUGUUCAAACAU
981
3157
AUGUUUGAACAACUUGGAA
1204

3153
UUUACCAAUCAUAUUAAUA
982
3153
UUUACCAAUCAUAUUAAUA
982
3175
UAUUAAUAUGAUUGGUAAA
1205

3171
ACAAUGAUGCUAUUUGCAA
983
3171
ACAAUGAUGCUAUUUGCAA
983
3193
UUGCAAAUAGCAUCAUUGU
1206

3189
AUUCCUGCUCCUAGGGGAG
984
3189
AUUCCUGCUCCUAGGGGAG
984
3211
CUCCCCUAGGAGCAGGAAU
1207

3207
GGGGAGAUAAGAAACCCUC
985
3207
GGGGAGAUAAGAAACCCUC
985
3229
GAGGGUUUCUUAUCUCCCC
1208

3225
CACUCUCUACAGGUUUGGG
986
3225
CACUCUCUACAGGUUUGGG
986
3247
CCCAAACCUGUAGAGAGUG
1209

3243
GUACAAGUGGCAACCUGCU
987
3243
GUACAAGUGGCAACCUGCU
987
3265
AGCAGGUUGCCACUUGUAC
1210

3261
UUCCAUGGCCGUGUAGAAG
988
3261
UUCCAUGGCCGUGUAGAAG
988
3283
CUUCUACACGGCCAUGGAA
1211

3279
GCAUGGUGCCCUGGCUUCU
989
3279
GCAUGGUGCCCUGGCUUCU
989
3301
AGAAGCCAGGGCACCAUGC
1212

3297
UCUGAGGAAGCUGGGGUUC
990
3297
UCUGAGGAAGCUGGGGUUC
990
3319
GAACCCCAGCUUCCUCAGA
1213

3315
CAUGACAAUGGCAGAUGUA
991
3315
CAUGACAAUGGCAGAUGUA
991
3337
UACAUCUGCCAUUGUCAUG
1214

3333
AAAGUUAUUCUUGAAGUCA
992
3333
AAAGUUAUUCUUGAAGUCA
992
3355
UGACUUCAAGAAUAACUUU
1215

3351
AGAUUGAGGCUGGGAGACA
993
3351
AGAUUGAGGCUGGGAGACA
993
3373
UGUCUCCCAGCCUCAAUCU
1216

3369
AGCCGUAGUAGAUGUUCUA
994
3369
AGCCGUAGUAGAUGUUCUA
994
3391
UAGAACAUCUACUACGGCU
1217

3387
ACUUUGUUCUGCUGUUCUC
995
3387
ACUUUGUUCUGCUGUUCUC
995
3409
GAGAACAGCAGAACAAAGU
1218

3405
CUAGAAAGAAUAUUUGGUU
996
3405
CUAGAAAGAAUAUUUGGUU
996
3427
AACCAAAUAUUCUUUCUAG
1219

3423
UUUCCUGUAUAGGAAUGAG
997
3423
UUUCCUGUAUAGGAAUGAG
997
3445
CUCAUUCCUAUACAGGAAA
1220

3441
GAUUAAUUCCUUUCCAGGU
998
3441
GAUUAAUUCCUUUCCAGGU
998
3463
ACCUGGAAAGGAAUUAAUC
1221

3459
UAUUUUAUAAUUCUGGGAA
999
3459
UAUUUUAUAAUUCUGGGAA
999
3481
UUCCCAGAAUUAUAAAAUA
1222

3477
AGCAAAACCCAUGCCUCCC
1000
3477
AGCAAAACCCAUGCCUCCC
1000
3499
GGGAGGCAUGGGUUUUGCU
1223

3495
CCCUAGCCAUUUUUACUGU
1001
3495
CCCUAGCCAUUUUUACUGU
1001
3517
ACAGUAAAAAUGGCUAGGG
1224

3513
UUAUCCUAUUUAGAUGGCC
1002
3513
UUAUCCUAUUUAGAUGGCC
1002
3535
GGCCAUCUAAAUAGGAUAA
1225

3531
CAUGAAGAGGAUGCUGUGA
1003
3531
CAUGAAGAGGAUGCUGUGA
1003
3553
UCACAGCAUCCUCUUCAUG
1226

3549
AAAUUCCCAACAAACAUUG
1004
3549
AAAUUCCCAACAAACAUUG
1004
3571
CAAUGUUUGUUGGGAAUUU
1227

3567
GAUGCUGACAGUCAUGCAG
1005
3567
GAUGCUGACAGUCAUGCAG
1005
3589
CUGCAUGACUGUCAGCAUC
1228

3585
GUCUGGGAGUGGGGAAGUG
1006
3585
GUCUGGGAGUGGGGAAGUG
1006
3607
CACUUCCCCACUCCCAGAC
1229

3603
GAUCUUUUGUUCCCAUCCU
1007
3603
GAUCUUUUGUUCCCAUCCU
1007
3625
AGGAUGGGAACAAAAGAUC
1230

3621
UCUUCUUUUAGCAGUAAAA
1008
3621
UCUUCUUUUAGCAGUAAAA
1008
3643
UUUUACUGCUAAAAGAAGA
1231

3639
AUAGCUGAGGGAAAAGGGA
1009
3639
AUAGCUGAGGGAAAAGGGA
1009
3661
UCCCUUUUCCCUCAGCUAU
1232

3657
AGGGAAAAGGAAGUUAUGG
1010
3657
AGGGAAAAGGAAGUUAUGG
1010
3679
CCAUAACUUCCUUUUCCCU
1233

3675
GGAAUACCUGUGGUGGUUG
1011
3675
GGAAUACCUGUGGUGGUUG
1011
3697
CAACCACCACAGGUAUUCC
1234

3693
GUGAUCCCUAGGUCUUGGG
1012
3693
GUGAUCCCUAGGUCUUGGG
1012
3715
CCCAAGACCUAGGGAUCAC
1235

3711
GAGCUCUUGGAGGUGUCUG
1013
3711
GAGCUCUUGGAGGUGUCUG
1013
3733
CAGACACCUCCAAGAGCUC
1236

3729
GUAUCAGUGGAUUUCCCAU
1014
3729
GUAUCAGUGGAUUUCCCAU
1014
3751
AUGGGAAAUCCACUGAUAC
1237

3747
UCCCCUGUGGGAAAUUAGU
1015
3747
UCCCCUGUGGGAAAUUAGU
1015
3769
ACUAAUUUCCCACAGGGGA
1238

3765
UAGGCUCAUUUACUGUUUU
1016
3765
UAGGCUCAUUUACUGUUUU
1016
3787
AAAACAGUAAAUGAGCCUA
1239

3783
UAGGUCUAGCCUAUGUGGA
1017
3783
UAGGUCUAGCCUAUGUGGA
1017
3805
UCCACAUAGGCUAGACCUA
1240

3801
AUUUUUUCCUAACAUACCU
1018
3801
AUUUUUUCCUAACAUACCU
1018
3823
AGGUAUGUUAGGAAAAAAU
1241

3819
UAAGCAAACCCAGUGUCAG
1019
3819
UAAGCAAACCCAGUGUCAG
1019
3841
CUGACACUGGGUUUGCUUA
1242

3837
GGAUGGUAAUUCUUAUUCU
1020
3837
GGAUGGUAAUUCUUAUUCU
1020
3859
AGAAUAAGAAUUACCAUCC
1243

3855
UUUCGUUCAGUUAAGUUUU
1021
3855
UUUCGUUCAGUUAAGUUUU
1021
3877
AAAACUUAACUGAACGAAA
1244

3873
UUCCCUUCAUCUGGGCACU
1022
3873
UUCCCUUCAUCUGGGCACU
1022
3895
AGUGCCCAGAUGAAGGGAA
1245

3891
UGAAGGGAUAUGUGAAACA
1023
3891
UGAAGGGAUAUGUGAAACA
1023
3913
UGUUUCACAUAUCCCUUCA
1246

3909
AAUGUUAACAUUUUUGGUA
1024
3909
AAUGUUAACAUUUUUGGUA
1024
3931
UACCAAAAAUGUUAACAUU
1247

3927
AGUCUUCAACCAGGGAUUG
1025
3927
AGUCUUCAACCAGGGAUUG
1025
3949
CAAUCCCUGGUUGAAGACU
1248

3945
GUUUCUGUUUAACUUCUUA
1026
3945
GUUUCUGUUUAACUUCUUA
1026
3967
UAAGAAGUUAAACAGAAAC
1249

GGAAAGCUUGAGUAAA
1027
3963
AUAGGAAAGCUUGAGUAAA
1027
3985
UUUACUCAAGCUUUCCUAU
1250

3981
AAUAAAUAUUGUCUUUUUG
1028
3981
AAUAAAUAUUGUCUUUUUG
1028
4003
CAAAAAGACAAUAUUUAUU
1251

3986
AUAUUGUCUUUUUGUAUGU
1029
3986
AUAUUGUCUUUUUGUAUGU
1029
4008
ACAUACAAAAAGACAAUAU
1252

The 3′-ends of the Upper sequence and the Lower sequence of the siNA construct can include an overhang sequence, for example about 1, 2, 3, or 4 nucleotides in length, preferably 2 nucleotides in length, wherein the overhanging sequence of the lower sequence is optionally complementary to a portion of the target sequence. The upper sequence is also referred to as the sense strand, whereas the lower sequence is also referred to as the antisense strand. The upper and lower sequences in the Table can further comprise a chemical modification having Formulae I-VII, such as exemplary siNA constructs shown in FIGS. 4 and 5, or having modifications described in Table IV or any combination thereof.

indicates data missing or illegible when filed

TABLE III

Interleukin and Interleukin receptor Synthetic Modified siNA constructs

Tar-

get

Seq

Seq

Pos
Target
ID
Cmpd#
Aliases
Sequence
ID

IL2RG

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 120U21 sense siNA
ACCACAGCUGAUUUCUUCCTT
1311

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 132U21 sense siNA
UUCUUCCUGACCACUAUGCTT
1312

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 140U21 sense siNA
GACCACUAUGCCCACUGACTT
1313

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 157U21 sense siNA
ACUCCCUCAGUGUUUCCACTT
1314

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 264U21 sense siNA
AACCUCACUCUGCAUUAUUTT
1315

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 304U21 sense siNA
AUAAAGUCCAGAAGUGCAGTT
1316

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 305U21 sense siNA
UAAAGUCCAGAAGUGCAGCTT
1317

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 346U21 sense siNA
UCACUUCUGGCUGUCAGUUTT
1318

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 138L21 antisense siNA
GGAAGAAAUCAGCUGUGGUTT
1319

(120C)

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 150L21 antisense siNA
GCAUAGUGGUCAGGAAGAATT
1320

(132C)

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 158L21 antisense siNA
GUCAGUGGGCAUAGUGGUCTT
1321

(140C)

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 175L21 antisense siNA
GUGGAAACACUGAGGGAGUTT
1322

(157C)

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 282L21 antisense siNA
AAUAAUGCAGAGUGAGGUUTT
1323

(264C)

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 322L21 antisense siNA
CUGCACUUCUGGACUUUAUTT
1324

(304C)

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 323L21 antisense siNA
GCUGCACUUCUGGACUUUATT
1325

(305C)

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 364L21 antisense siNA
AACUGACAGCCAGAAGUGATT
1326

(346C)

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 120U21 sense siNA stab04
B AccAcAGcuGAuuucuuccTT B
1327

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 132U21 sense siNA stab04
B uucuuccuGAccAcuAuGcTT B
1328

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 140U21 sense siNA stab04
B GAccAcuAuGcccAcuGAcTT B
1329

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 157U21 sense siNA stab04
B AcucccucAGuGuuuccAcTT B
1330

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 264U21 sense siNA stab04
B AAccucAcucuGcAuuAuuTT B
1331

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 304U21 sense siNA stab04
B AuAAAGuccAGAAGuGcAGTT B
1332

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 305U21 sense siNA stab04
B uAAAGuccAGAAGuGcAGcTT B
1333

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 346U21 sense siNA stab04
B ucAcuucuGGcuGucAGuuTT B
1334

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 138L21 antisense siNA
GGAAGAAAucAGcuGuGGuTsT
1335

(120C) stab05

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 150L21 antisense siNA
GcAuAGuGGucAGGAAGAATsT
1336

(132C) stab05

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 158L21 antisense siNA
GucAGuGGGcAuAGuGGuCTsT
1337

(140C) stab05

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 175L21 antisense siNA
GuGGAAAcAcuGAGGGAGuTsT
1338

(157C) stab05

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 282L21 antisense siNA
AAuAAuGcAGAGuGAGGuuTsT
1339

(264C) stab05

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 322L21 antisense siNA
cuGcAcuucuGGAcuuUAUTsT
1340

(304C) stab05

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 323L21 antisense siNA
GcuGcAcuucuGGAcuuuATsT
1341

(305C) stab05

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 364L21 antisense siNA
AAcuGAcAGccAGAAGuGATsT
1342

(346C) stab05

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 120U21 sense siNA stab07
B AccAcAGcuGAuuucuuccTT B
1343

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 132U21 sense siNA stab07
B uucuuccuGAccAcuAuGcTT B
1344

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 140U21 sense siNA stab07
B GAccAcuAuGcccAcuGAcTT B
1345

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 157U21 sense siNA stab07
B AcucccucAGuGuuuccAcTT B
1346

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 264U21 sense siNA stab07
B AAccucAcucuGcAuuAuuTT B
1347

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 304U21 sense siNA stab07
B AuAAAGuccAGAAGuGcAGTT B
1348

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 305U21 sense siNA stab07
B uAAAGuccAGAAGuGcAGcTT B
1349

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 346U21 sense siNA stab07
B ucAcuucuGGcuGucAGuuTT B
1350

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 138L21 antisense siNA

GGAAGAAAucAGcuGuGGuTsT
1351

(120C) stab11

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 150L21 antisense siNA

GcAuAGuGGucAGGAAGAATsT
1352

(132C) stab11

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 158L21 antisense siNA

GucAGuGGGcAuAGuGGucTsT
1353

(140C) stab11

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 175L21 antisense siNA

GuGGAAAcAcuGAGGGAGuTsT
1354

(157C) stab11

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 282L21 antisense siNA

AAuAAuGcAGAGuGAGGuuTsT
1355

(264C) stab11

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 322L21 antisense siNA
cuGcAcuucuGGAcuuuAuTsT
1356

(304C) stab11

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 323L21 antisense siNA

GcuGcAcuucuGGAcuuuATsT
1357

(305C) stab11

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 364L21 antisense siNA

AAcuGAcAGccAGAAGuGATsT
1358

(346C) stab11

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 120U21 sense siNA stab18
B AccAcAGcuGAuuucuuccTT B
1359

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 132U21 sense siNA stab18
B uucuuccuGAccAcuAuGcTT B
1360

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 140U21 sense siNA stab18
B GAccAcuAuGcccAcuGAcTT B
1361

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 157U21 sense siNA stab18
B AcucccucAGuGuuuccAcTT B
1362

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 264U21 sense siNA stab18
B AAccucAcucuGcAuuAuuTT B
1363

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 304U21 sense siNA stab18
B AuAAAGuccAGAAGuGcAGTT B
1364

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 305U21 sense siNA stab18
B uAAAGuccAGAAGuGcAGcTT B
1365

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 346U21 sense siNA stab18
B ucAcuucuGGcuGucAGuuTT B
1366

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 138L21 antisense siNA

GGAAGAAAucAGcuGuGGuTsT
1367

(120C) stab08

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 150L21 antisense siNA

GcAuAGuGGucAGGAAGAATsT
1368

(132C) stab08

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 158L21 antisense siNA

GucAGuGGGcAuAGuGGuGTsT
1369

(140C) stab08

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 175L21 antisense siNA

GuGGAAAcAcuGAGGGAGuTsT
1370

(157C) stab08

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 282L21 antisense siNA

AAuAAuGcAGAGuGAGGuuTsT
1371

(264C) stab08

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 322L21 antisense siNA
cuGcAcuucuGGAcuuuAuTsT
1372

(304C) stab08

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 323L21 antisense siNA

GcuGcAcuucuGGAcuuuATsT
1373

(305C) stab08

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 364L21 antisense siNA

AAcuGAcAGccAGAAGuGATsT
1374

(346C) stab08

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 120U21 sense siNA stab09
B ACCACAGCUGAUUUCUUCCTT B
1375

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 132U21 sense siNA stab09
B UUCUUCCUGACCACUAUGCTT B
1376

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 140U21 sense siNA stab09
B GACCACUAUGCCCACUGACTT B
1377

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 157U21 sense siNA stab09
B ACUCCCUCAGUGUUUCCACTT B
1378

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 264U21 sense siNA stab09
B AACCUCACUCUGCAUUAUUTT B
1379

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 304U21 sense siNA stab09
B AUAAAGUCCAGAAGUGCAGTT B
1380

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 305U21 sense siNA stab09
B UAAAGUCCAGAAGUGCAGCTT B
1381

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 346U21 sense siNA stab09
B UCACUUCUGGCUGUCAGUUTT B
1382

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 138L21 antisense siNA
GGAAGAAAUCAGCUGUGGUTsT
1383

(120C) stab10

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 150L21 antisense siNA
GCAUAGUGGUCAGGAAGAATsT
1384

(132C) stab10

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 158L21 antisense siNA
GUCAGUGGGCAUAGUGGUCTsT
1385

(140C) stab10

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 175L21 antisense siNA
GUGGAAACACUGAGGGAGUTsT
1386

(157C) stab10

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 282L21 antisense siNA
AAUAAUGCAGAGUGAGGUUTsT
1387

(264C) stab10

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 322L21 antisense siNA
CUGCACUUCUGGACUUUAUTsT
1388

(304C) stab10

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 323L21 antisense siNA
GCUGCACUUCUGGACUUUATsT
1389

(305C) stab10

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 364L21 antisense siNA
AACUGACAGCCAGAAGUGATsT
1390

(346C) stab10

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 138L21 antisense siNA
GGAAGAAAucAGCuGuGGuTT B
1391

(120C) stab19

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 150L21 antisense siNA
GcAuAGuGGucAGGAAGAATT B
1392

(132C) stab19

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 158L21 antisense siNA
GucAGuGGGCAuAGuGGucTT B
1393

(140C) stab19

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 175L21 antisense siNA
GuGGAAACAcuGAGGGAGuTT B
1394

(157C) stab19

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 282L21 antisense siNA
AAuAAuGCAGAGuGAGGuUTT B
1395

(264C) stab19

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 322L21 antisense siNA
cuGcAcuucuGGAcuuuAuTT B
1396

(304C) stab19

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 323L21 antisense siNA
GcuGcAcuucuGGAcuuuATT B
1397

(305C) stab19

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 364L21 antisense siNA
AAcuGAcAGcCAGAAGuGATT B
1398

(346C) stab19

118
ACACCACAGCUGAUUUCUUCCUG
1253

IL2RG: 138L21 antisense siNA
GGAAGAAAUCAGCUGUGGUTT B
1399

(120C) stab22

130
AUUUCUUCCUGACCACUAUGCCC
1254

IL2RG: 150L21 antisense siNA
GCAUAGUGGUCAGGAAGAATT B
1400

(132C) stab22

138
CUGACCACUAUGCCCACUGACUC
1255

IL2RG: 158L21 antisense siNA
GUCAGUGGGCAUAGUGGUCTT B
1401

(140C) stab22

155
UGACUCCCUCAGUGUUUCCACUC
1256

IL2RG: 175L21 antisense siNA
GUGGAAACACUGAGGGAGUTT B
1402

(157C) stab22

262
CCAACCUCACUCUGCAUUAUUGG
1257

IL2RG: 282L21 antisense siNA
AAUAAUGCAGAGUGAGGUUTT B
1403

(264C) stab22

302
UGAUAAAGUCCAGAAGUGCAGCC
1258

IL2RG: 322L21 antisense siNA
CUGCACUUCUGGACUUUAUTT B
1404

(304C) stab22

303
GAUAAAGUCCAGAAGUGCAGCCA
1259

IL2RG: 323L21 antisense siNA
GCUGCACUUCUGGACUUUATT B
1405

(305C) stab22

344
AAUCACUUCUGGCUGUCAGUUGC
1260

IL2RG: 364L21 antisense siNA
AACUGACAGCCAGAAGUGATT B
1406

(346C) stab22

IL4

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 489U21 sense siNA
GCCUCACAGAGCAGAAGACTT
1407

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 491U21 sense siNA
CUCACAGAGCAGAAGACUCTT
1408

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 518U21 sense siNA
GAGUUGACCGUAACAGACATT
1409

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 528U21 sense siNA
UAACAGACAUCUUUGCUGCTT
1410

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 547U21 sense siNA
CUCCAAGAACACAACUGAGTT
1411

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 608U21 sense siNA
UACAGCCACCAUGAGAAGGTT
1412

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 730U21 sense siNA
GAAUUCCUGUCCUGUGAAGTT
1413

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 747U21 sense siNA
AGGAAGCCAACCAGAGUACTT
1414

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 507L21 antisense siNA
GUCUUCUGCUCUGUGAGGCTT
1415

(489C)

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 509L21 antisense siNA
GAGUCUUCUGCUCUGUGAGTT
1416

(491C)

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 536L21 antisense siNA
UGUCUGUUACGGUCAACUCTT
1417

(518C)

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 546L21 antisense siNA
GCAGCAAAGAUGUCUGUUATT
1418

(528C)

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 565L21 antisense siNA
CUCAGUUGUGUUCUUGGAGTT
1419

(547C)

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 626L21 antisense siNA
CCUUCUCAUGGUGGCUGUATT
1420

(608C)

728
UUGAAUUGCUGUCCUGUGAAGGA
1275

IL4: 748L21 antisense siNA
CUUCACAGGACAGGAAUUCTT
1421

(730C)

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 765L21 antisense siNA
GUACUCUGGUUGGCUUCCUTT
1422

(747C)

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 489U21 sense siNA stab04
B GccucAcAGAGcAGAAGACTT B
1423

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 491U21 sense siNA stab04
B cucAcAGAGcAGAAGAcuc1T B
1424

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 518U21 sense siNA stab04
B GAGuuGAccGuAAcAGAcATT B
1425

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 528U21 sense siNA stab04
B uAAcAGAcAucuuuGcuGcTT B
1426

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 547U21 sense siNA stab04
B cuccAAGAAcACAACuGAGTT B
1427

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 608U21 sense siNA stab04
B uAcAGccAccAuGAGAAGGTT B
1428

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 730U21 sense siNA stab04
B GAAuuccuGuccuGuGAAGTT B
1429

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 747U21 sense siNA stab04
B AGGAAGccAAccAGAGuAcTT B
1430

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 507L21 antisense siNA
GucuucuGcucuGuGAGGcTsT
1431

(489C) stab05

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 509L21 antisense siNA
GAGucuucuGcucuGuGAGTsT
1432

(491C) stab05

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 536L21 antisense siNA
uGucuGuuAcGGucMcuoTsT
1433

(518C) stab05

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 546L21 antisense siNA
GcAGcAAAGAUGuCuGuuATsT
1434

(528C) stab05

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 565L21 antisense siNA
cucAGuuGuGuucuuGGAGTsT
1435

(547C) stab05

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 626L21 antisense siNA
ccuucucAuGGuGGcuGuATsT
1436

(608C) stab05

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 748L21 antisense siNA
cuucAcAGGAcAGGAAuucTsT
1437

(730C) stab05

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 765L21 antisense siNA
GuAcucuGGuuGgcuuccuTsT
1438

(747C) stab05

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 489U21 sense siNA stab07
B GccucAcAGAGcAGAAGAcTT B
1439

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 491U21 sense siNA stab07
B cucAcAGAGcAGAAGAcucTT B
1440

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 518U21 sense siNA stab07
B GAGuuGAccGuAAcAGAcATT B
1441

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 528U21 sense siNA stab07
B uAAcAGAcAucuuuGcuGcTT B
1442

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 547U21 sense siNA stab07
B cuccAAGAAcAcAACuGAGTT B
1443

606
UCUACAGCCAGCAUGAGAAGGAC
1274

IL4: 608U21 sense siNA stab07
B uAcAGccAccAuGAGAAGGTT B
1444

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 730U21 sense siNA stab07
B GAAuuccuGuccuGuGAAGTT B
1445

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 747U21 sense siNA stab07
B AGGAAGccAAccAGAGuAcTT B
1446

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 507L21 antisense siNA

GucuucuGcucuGuGAGGcTsT
1447

(489C) stab11

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 509L21 antisense siNA

GAGucuucuGcucuGuGAGTsT
1448

(491C) stab11

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 536L21 antisense siNA
uGucuGuuAcGGucAAcucTsT
1449

(518C) stab11

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 546L21 antisense siNA

GcAGcAAAGAuGucuGuuATsT
1450

(528C) stab11

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 565L21 antisense siNA
cucAGuuGuGuucuuGGAGTsT
1451

(547C) stab11

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 626L21 antisense siNA
ccuucucAuGGuGGcuGuATsT
1452

(608C) stab11

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 748L21 antisense siNA
cuucAcAGGAcAGGAAuucTsT
1453

(730C) stab11

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 765L21 antisense siNA

GuAcucuGGuuGGcuuccuTsT
1454

(747C) stab11

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 489U21 sense siNA stab18
B GccucAcAGAGcAGAAGAcTT B
1455

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 491U21 sense siNA stab18
B cucAcAGAGcAGAAGAcucTT B
1456

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 518U21 sense siNA stab18
B GAGuuGAccGuAAcAGAcATT B
1457

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 528U21 sense siNA stab18
B uAAcAGAcAucuuuGcuGcTT B
1458

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 547U21 sense siNA stab18
B cuccAAGAAcAcAAcuGAGTT B
1459

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 608U21 sense siNA stab18
B uAcAGccAccAuGAGAAGGTT B
1460

728
UUGAAUUGCUGUCCUGUGAAGGA
1275

IL4: 730U21 sense siNA stab18
B GAAuuccuGuccuGuGAAGTT B
1461

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 747U21 sense siNA stab18
B AGGAAGccAAccAGAGuAcTT B
1462

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 507L21 antisense siNA

GucuucuGcucuGuGAGGcTsT
1463

(489C) stab08

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 509L21 antisense siNA

GAGucuucuGcucuGuGAGTsT
1464

(491C) stab08

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 536L21 antisense siNA
uGucuGuuAcGGucAAcucTsT
1465

(518C) stab08

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 546L21 antisense siNA

GcAGcAAAGAuGucuGuuATsT
1466

(528C) stab08

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 565L21 antisense siNA
cucAGuuGuGuucuuGGAGTsT
1467

(547C) stab08

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 626L21 antisense siNA
ccuucucAuGGuGGcuGuATsT
1468

(608C) stab08

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 748L21 antisense siNA
cuucAcAGGAcAGGAAuucTsT
1469

(730C) stab08

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 765L21 antisense siNA

GuAcucuGGuuGGcuuccuTsT
1470

(747C) stab08

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 489U21 sense siNA stab09
B GCCUCACAGAGCAGAAGACTT B
1471

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 491U21 sense siNA stab09
B CUCACAGAGCAGAAGACUCTT B
1472

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 518U21 sense siNA stab09
B GAGUUGACCGUAACAGACATT B
1473

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 528U21 sense siNA stab09
B UAACAGACAUCUUUGCUGCTT B
1474

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 547U21 sense siNA stab09
B CUCCAAGAACACAACUGAGTT B
1475

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 608U21 sense siNA stab09
B UACAGCCACCAUGAGAAGGTT B
1476

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 730U21 sense siNA stab09
B GAAUUCCUGUCCUGUGAAGTT B
1477

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 747U21 sense siNA stab09
B AGGAAGCCAACCAGAGUACTT B
1478

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 507L21 antisense siNA
GUCUUCUGCUCUGUGAGGCTsT
1479

(489C) stab10

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 509L21 antisense siNA
GAGUCUUCUGCUCUGUGAGTsT
1480

(491C) stab10

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 536L21 antisense siNA
UGUCUGUUACGGUCAACUCTsT
1481

(518C) stab10

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 546L21 antisense siNA
GCAGCAAAGAUGUCUGUUATsT
1482

(528C) stab10

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 565L21 antisense siNA
CUCAGUUGUGUUCUUGGAGTsT
1483

(547C) stab10

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 626L21 antisense siNA
CCUUCUCAUGGUGGCUGUATsT
1484

(608C) stab10

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 748L21 antisense siNA
CUUCACAGGACAGGAAUUCTsT
1485

(730C) stabl 0

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 765L21 antisense siNA
GUACUCUGGUUGGCUUCCUTsT
1486

(747C) stab10

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 507L21 antisense siNA
GucuucuGcucuGuGAGGcTT B
1487

(489C) stab19

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 509L21 antisense siNA
GAGucuucuGcucuGuGAGTT B
1488

(491C) stab19

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 536L21 antisense siNA
uGucuGuuAcGGucAAcucTT B
1489

(518C) stab19

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 546L21 antisense siNA
GcAGcAAAGAuGucuGuuATT B
1490

(528C) stab19

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 565L21 antisense siNA
cucAGuuGuGuucuuGGAGTT B
1491

(547C) stab19

606
UCUACAGCCACCAUGAGAAGGA
1274

IL4: 626L21 antisense siNA
ccuucucAuGGuGGcuGuATT B
1492

(608C) stab19

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 748L21 antisense siNA
cuucAcAGGAcAGGAAuucTT B
1493

(730C) stab19

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 765L21 antisense siNA
GuAcucuGGuuGGcuuccuTT B
1494

(747C) stab19

487
CAGCCUCACAGAGCAGAAGACUC
1269

IL4: 507L21 antisense siNA
GUCUUCUGCUCUGUGAGGCTT B
1495

(489C) stab22

489
GCCUCACAGAGCAGAAGACUCUG
1270

IL4: 509L21 antisense siNA
GAGUCUUCUGCUCUGUGAGTT B
1496

(491C) stab22

516
CCGAGUUGACCGUAACAGACAUC
1271

IL4: 536L21 antisense siNA
UGUCUGUUACGGUCAACUCTT B
1497

(518C) stab22

526
CGUAACAGACAUCUUUGCUGCCU
1272

IL4: 546L21 antisense siNA
GCAGCAAAGAUGUCUGUUATT B
1498

(528C) stab22

545
GCCUCCAAGAACACAACUGAGAA
1273

IL4: 565L21 antisense siNA
CUCAGUUGUGUUCUUGGAGTT B
1499

(547C) stab22

606
UCUACAGCCACCAUGAGAAGGAC
1274

IL4: 626L21 antisense siNA
CCUUCUCAUGGUGGCUGUATT B
1500

(608C) stab22

728
UUGAAUUCCUGUCCUGUGAAGGA
1275

IL4: 748L21 antisense siNA
CUUCACAGGACAGGAAUUCTT B
1501

(730C) stab22

745
GAAGGAAGCCAACCAGAGUACGU
1276

IL4: 765L21 antisense siNA
GUACUCUGGUUGGCUUCCUTT B
1502

(747C) stab22

IL4R

469
CUAUACACUGGACCUGUGGGCUG
1277

IL4R: 471U21 sense siNA
AUACACUGGACCUGUGGGCTT
1503

551
CCAGGAAACCUGACAGUUCACAC
1278

IL4R: 553U21 sense siNA
AGGAAACCUGACAGUUCACTT
1504

1119
AGCACAACAUGAAAAGGGAUGAA
1279

IL4R: 1121U21 sense siNA
CACAACAUGAAAAGGGAUGTT
1505

1120
GCACAACAUGAAAAGGGAUGAAG
1280

IL4R: 1122U21 sense siNA
ACAACAUGAAAAGGGAUGATT
1506

1132
AAGGGAUGAAGAUCCUCACAAGG
1281

IL4R: 1134U21 sense siNA
GGGAUGAAGAUCCUCACAATT
1507

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282

IL4R: 3132U21 sense siNA
GGGAAAUCGAUGAGAAAUUTT
1508

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283

IL4R: 3133U21 sense siNA
GGAAAUCGAUGAGAAAUUGTT
1509

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284

IL4R: 3171U21 sense siNA
AUUGCCUAGAGGUGCUCAUTT
1510

469
CUAUACACUGGACCUGUGGGCUG
1277

IL4R: 489L21 antisense siNA
GCCCACAGGUCCAGUGUAUTT
1511

(471C)

551
CCAGGAAACCUGACAGUUCACAC
1278

IL4R: 571L21 antisense siNA
GUGAACUGUCAGGUUUCCUTT
1512

(553C)

1119
AGCACAACAUGAAAAGGGAUGAA
1279

IL4R: 1139L21 antisense siNA
CAUCCCUUUUCAUGUUGUGTT
1513

(1121C)

1120
GCACAACAUGAAAAGGGAUGAAG
1280

IL4R: 1140L21 antisense siNA
UCAUCCCUUUUCAUGUUGUTT
1514

1122C

1132
AAGGGAUGAAGAUCCUCACAAGG
1281

IL4R: 1152L21 antisense siNA
UUGUGAGGAUCUUCAUCCCTT
1515

(1134C)

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282

IL4R: 3150L21 antisense siNA
AAUUUCUCAUCGAUUUCCCTT
1516

(3132C)

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283

IL4R: 3151L21 antisense siNA
CAAUUUCUCAUCGAUUUCCTT
1517

(3133C)

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284

IL4R: 3189L21 antisense siNA
AUGAGCACCUCUAGGCAAUTT
1518

(3171C)

469
CUAUACACUGGACCUGUGGGCUG
1277

IL4R: 471U21 sense siNA stab04
B AuAcAcuGGAccuGuGGGcTT B
1519

551
CCAGGAAACCUGACAGUUCACAC
1278

IL4R: 553U21 sense siNA stab04
B AGGAAAccuGAcAGuucAcTT B
1520

1119
AGCACAACAUGAAAAGGGAUGAA
1279

IL4R: 1121U21 sense siNA stab04
B cAcAAcAuGAAAAGGGAuGTT B
1521

1120
GCACAACAUGAAAAGGGAUGAAG
1280

IL4R: 1122U21 sense siNA stab04
B AcAAcAuGAAAAGGGAuGATT B
1522

1132
AAGGGAUGAAGAUCCUCACAAGG
1281

IL4R: 1134U21 sense siNA stab04
B GGGAuGAAGAuccucAcAATT B
1523

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282

IL4R: 3132U21 sense siNA stab04
B GGGAAAucGAuGAGAAAuu1T B
1524

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283

IL4R: 3133U21 sense siNA stab04
B GGAAAucGAuGAGAAAuuGTT B
1525

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284

IL4R: 3171U21 sense siNA stab04
B AuuGccuAGAGGuGcucAuTT B
1526

469
CUAUACACUGGACCUGUGGGCUG
1277

IL4R: 489L21 antisense siNA
GcccAcAGGuccAGuGuAuTsT
1527

(471C) stab05

551
CCAGGAAACCUGACAGUUCACAC
1278

IL4R: 571L21 antisense siNA
GuGAAcuGucAGGuuuccuTsT
1528

(553C) stab05

1119
AGCACAACAUGAAAAGGGAUGAA
1279

IL4R: 1 139L21 antisense siNA
cAucccuuuucAuGuuGuGTsT
1529

(1121C) stab05

1120
GCACAACAUGAAAAGGGAUGAAG
1280

IL4R: 1 140L21 antisense siNA
ucAucccuuuucAuGuuGulsT
1530

(1122C) stab05

1132
AAGGGAUGAAGAUCCUCACAAGG
1281

IL4R: 1 152L21 antisense siNA
uuGuGAGGAucuucAucccTsT
1531

(1134C) stab05

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282

IL4R: 3150L21 antisense siNA
AAuuucucAucGAuuucccTsT
1532

(3132C) stab05

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283

IL4R: 3151 L21 antisense siNA
cAAuuucucAucGAuuuccTsT
1533

(3133C) stab05

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284

IL4R: 3189L21 antisense siNA
AuGAGcAccucuAGGcAAuTsT
1534

(3171C) stab05

469
CUAUACACUGGACCUGUGGGCUG
1277

IL4R: 471U21 sense siNA stab07
B AuAcAcuGGAccuGuGGGcTT B
1535

551
CCAGGAAACCUGACAGUUCACAC
1278

IL4R: 553U21 sense siNA stab07
B AGGAAAccuGAcAGuucAcTT B
1536

1119
AGCACAACAUGAAAAGGGAUGAA
1279

IL4R: 1121U21 sense siNA stab07
B cAcAAcAuGAAAAGGGAuGTT B
1537

1120
GCACAACAUGAAAAGGGAUGAAG
1280

IL4R: 1122U21 sense siNA stab07
B AcAAcAuGAAAAGGGAuGATT B
1538

1132
AAGGGAUGAAGAUCCUCACAAGG
1281

IL4R: 1134U21 sense siNA stab07
B GGGAuGAAGAuccucAcAATT B
1539

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282

IL4R: 3132U21 sense siNA stab07
B GGGAAAucGAuGAGAAAuuTT B
1540

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283

IL4R: 3133U21 sense siNA stab07
B GGAAAucGAuGAGAAAuuGTT B
1541

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284

IL4R: 3171U21 sense siNA stab07
B AuuGccuAGAGGuGcucAuTT B
1542

469
CUAUACACUGGACCUGUGGGCUG
1277

IL4R: 489L21 antisense siNA

GcccAcAGGuccAGuGuAuTsT
1543

(471C) stab11

551
CCAGGAAACCUGACAGUUCACAC
1278

IL4R: 571L21 antisense siNA

GuGAAcuGucAGGuuuccuTsT
1544

(553C) stab11

1119
AGCACAACAUGAAAAGGGAUGAA
1279

IL4R: 1139L21 antisense siNA
cAucccuuuucAuGuuGuGTsT
1545

(1121C) stab11

1120
GCACAACAUGAAAAGGGAUGAAG
1280

IL4R: 1140L21 antisense siNA
ucAucccuuuucAuGuuGuTsT
1546

(1122C) stab11

1132
AAGGGAUGAAGAUCCUCACAAGG
1281

IL4R: 1152L21 antisense siNA
uuGuGAGGAucuucAucccTsT
1547

(1134C) stab11

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282

IL4R: 3150L21 antisense siNA

AAuuucucAucGAuuucccTsT
1548

(3132C) stab11

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283

IL4R: 3151L21 antisense siNA
cAAuuucucAucGAuuuccTsT
1549

(3133C) stab11

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284

IL4R: 3189L21 antisense siNA

AuGAGcAccucuAGGcAAuTsT
1550

(3171C) stab11

469
CUAUACACUGGACCUGUGGGCUG
1277

IL4R: 471U21 sense siNA stab18
B AuAcAcuGGAccuGuGGGcTT B
1551

551
CCAGGAAACCUGACAGUUCACAC
1278

IL4R: 553U21 sense siNA stab18
B AGGAAAccuGAcAGuucAcTT B
1552

1119
AGCACAACAUGAAAAGGGAUGAA
1279

IL4R: 1121U21 sense siNA stab18
B cAcAAcAuGAAAAGGGAuGTT B
1553

1120
GCACAACAUGAAAAGGGAUGAAG
1280

IL4R: 1122U21 sense siNA stab18
B AcAAcAuGAAAAGGGAuGATT B
1554

1132
AAGGGAUGAAGAUCCUCACAAGG
1281

IL4R: 1134U21 sense siNA stab18
B GGGAuGAAGAuccucAcAATT B
1555

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282

IL4R: 3132U21 sense siNA stab18
B GGGAAAucGAuGAGAAAuuTT B
1556

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283

IL4R: 3133U21 sense siNA stab18
B GGAAAucGAuGAGAAAuuGTT B
1557

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284

IL4R: 3171U21 sense siNA stab18
B AuuGccuAGAGGuGcucAuTT B
1558

469
CUAUACACUGGACCUGUGGGCUG
1277

IL4R: 489L21 antisense siNA

GcccAcAGGuccAGuGuAuTsT
1559

(471C) stab08

551
CCAGGAAACCUGACAGUUCACAC
1278

IL4R: 571L21 antisense siNA

GuGAAcuGucAGGuuuccuTsT
1560

(553C) stabOB

1119
AGCACAACAUGAAAAGGGAUGAA
1279

IL4R: 1139L21 antisense siNA
cAucccuuuucAuGuuGuGTsT
1561

(1121C) stab08

1120
GCACAACAUGAAAAGGGAUGAAG
1280

IL4R: 1140L21 antisense siNA
ucAucccuuuucAuGuuGuTsT
1562

(1122C) stab08

1132
AAGGGAUGAAGAUCCUCACAAGG
1281

IL4R: 1152L21 antisense siNA
uuGuGAGGAucuucAucccTsT
1563

(1134C) stab08

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282

IL4R: 3150L21 antisense siNA

AAuuucucAucGAuuucccTsT
1564

(3132C) stabC8

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283

IL4R: 3151L21 antisense siNA
cAAuuucucAucGAuuuccTsT
1565

(3133C) stab08

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284

IL4R: 3189L21 antisense siNA

AuGAGcAccucuAGGcAAuTsT
1566

(3171C) stab08

469
CUAUACACUGGACCUGUGGGCUG
1277
36729
IL4R: 471U21 sense siNA stab09
B AUACACUGGACCUGUGGGCTT B
1567

551
CCAGGAAACCUGACAGUUCACAC
1278
36730
IL4R: 553U21 sense siNA stab09
B AGGAAACCUGACAGUUCACTT B
1568

1119
AGCACAACAUGAAAAGGGAUGAA
1279
36731
IL4R: 1121U21 sense siNA stab09
B CACAACAUGAAAAGGGAUGTT B
1569

1120
GCACAACAUGAAAAGGGAUGAAG
1280
36732
IL4R: 1122U21 sense siNA stab09
B ACAACAUGAAAAGGGAUGATT B
1570

1132
AAGGGAUGAAGAUCCUCACAAGG
1281
36733
IL4R: 1134U21 sense siNA stab09
B GGGAUGAAGAUCCUCACAATT B
1571

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282
36734
IL4R: 3132U21 sense siNA stab09
B GGGAAAUCGAUGAGAAAUUTT B
1572

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283
36735
IL4R: 3133U21 sense siNA stab09
B GGAAAUCGAUGAGAAAUUGTT B
1573

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284
36736
IL4R: 3171U21 sense siNA stab09
B AUUGCCUAGAGGUGCUCAUTT B
1574

469
CUAUACACUGGACCUGUGGGCUG
1277

IL4R: 489L21 antisense siNA
GCCCACAGGUCCAGUGUAUTsT
1575

(471C) stab10

551
CCAGGAAACCUGACAGUUCACAC
1278

IL4R: 571L21 antisense siNA
GUGAACUGUCAGGUUUCCUTsT
1576

(553C) stab10

1119
AGCACAACAUGAAAAGGGAUGAA
1279

IL4R: 1139L21 antisense siNA
CAUCCCUUUUCAUGUUGUGTsT
1577

(1121C) stab10

1120
GCACAACAUGAAAAGGGAUGAAG
1280

IL4R: 1140L21 antisense siNA
UCAUCCCUUUUCAUGUUGUTsT
1578

(1122C) stab10

1132
AAGGGAUGAAGAUCCUCACAAGG
1281

IL4R: 1152L21 antisense siNA
UUGUGAGGAUCUUCAUCCCTsT
1579

(1134C) stab10

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282

IL4R: 3150L21 antisense siNA
AAUUUCUCAUCGAUUUCCCTsT
1580

(3132C) stab10

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283

IL4R: 3151L21 antisense siNA
CAAUUUCUCAUCGAUUUCCTsT
1581

(3133C) stab10

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284

IL4R: 3189L21 antisense siNA
AUGAGCACCUCUAGGCAAUTsT
1582

(3171C) stab10

469
CUAUACACUGGACCUGUGGGCUG
1277
36737
IL4R: 489L21 antisense siNA
GcccAcAGGuccAGuGuAuTT B
1583

(471C) stab19

551
CCAGGAAACCUGACAGUUCACAC
1278
36738
IL4R: 571L21 antisense siNA
GuGAAcuGucAGGuuuccuTT B
1584

(553C) stab19

1119
AGCACAACAUGAAAAGGGAUGAA
1279
36739
IL4R: 1139L21 antisense siNA
cAucccuuuucAuGuuGuGTT B
1585

(1121C) stab19

1120
GCACAACAUGAAAAGGGAUGAAG
1280
36740
IL4R: 1140L21 antisense siNA
ucAucccuuuucAuGuuGuTT B
1586

(1122C) stab19

1132
AAGGGAUGAAGAUCCUCACAAGG
1281
36741
IL4R: 1152L21 antisense siNA
uuGuGAGGAucuucAucccTT B
1587

(1134C) stab19

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282
36742
IL4R: 3150L21 antisense siNA
AAuuucucAucGAuuucccTT B
1588

(3132C) stab19

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283
36743
IL4R: 3151L21 antisense siNA
cAAuuucucAucGAuuuccTT B
1589

(3133C) stab19

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284
36744
IL4R: 3189L21 antisense siNA
AuGAGcAccucuAGGcAAuTT B
1590

(3171C) stab19

469
CUAUACACUGGACCUGUGGGCUG
1277
36745
IL4R: 489L21 antisense siNA
GCCCACAGGUCCAGUGUAUTT B
1591

(471C) stab22

551
CCAGGAAACCUGACAGUUCACAC
1278
36746
IL4R: 571L21 antisense siNA
GUGAACUGUCAGGUUUCCUTT B
1592

(553C) stab22

1119
AGCACAACAUGAAAAGGGAUGAA
1279
36747
IL4R: 1139L21 antisense siNA
CAUCCCUUUUCAUGUUGUGTT B
1593

(1121C) stab22

1120
GCACAACAUGAAAAGGGAUGAAG
1280
36748
IL4R: 1140L21 antisense siNA
UCAUCCCUUUUCAUGUUGUTT B
1594

(1122C) stab22

1132
AAGGGAUGAAGAUCCUCACAAGG
1281
36749
IL4R: 1152L21 antisense siNA
UUGUGAGGAUCUUCAUCCCTT B
1595

(1134C) stab22

3130
UUGGGAAAUCGAUGAGAAAUUGA
1282
36750
IL4R: 3150L21 antisense siNA
AAUUUCUCAUCGAUUUCCCTT B
1596

(3132C) stab22

3131
UGGGAAAUCGAUGAGAAAUUGAA
1283
36751
IL4R: 3151L21 antisense siNA
CAAUUUCUCAUCGAUUUCCTT B
1597

(3133C) stab22

3169
UCAUUGCCUAGAGGUGCUCAUUC
1284
36752
IL4R: 3189L21 antisense siNA
AUGAGCACCUCUAGGCAAUTT B
1598

(3171C) stab22

IL13

391
CCCAGUUUGUAAAGGACCUGCUC
1285

IL13: 393U21 sense siNA
CAGUUUGUAAAGGACCUGCTT
1599

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 799U21 sense siNA
CUUCACACACAGGCAACUGTT
1600

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 834U21 sense siNA
AGGCACACUUCUUCUUGGUTT
1601

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 913U21 sense siNA
GACUGUGGCUGCUAGCACUTT
1602

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 965U21 sense siNA
CACUAAAGCAGUGGACACCTT
1603

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 967U21 sense siNA
CUAAAGCAGUGGACACCAGTT
1604

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 970U21 sense siNA
AAGCAGUGGACACCAGGAGTT
1605

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1193U21 sense siNA
AAGGGUACCUUGAACACUGTT
1606

3910
CCAGUUUGUAAAGGACCUGCUC
1285

IL13: 411L21 antisense siNA
GCAGGUCCUUUACAAACUGTT
1607

(393C)

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 817L21 antisense siNA
CAGUUGCCUGUGUGUGAAGTT
1608

(799C)

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 852L21 antisense siNA
ACCAAGAAGAAGUGUGCCUTT
1609

(834C)

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 931L21 antisense siNA
AGUGCUAGCAGCCACAGUCTT
1610

(913C)

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 983L21 antisense siNA
GGUGUCCACUGCUUUAGUGTT
1611

(965C)

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 985L21 antisense siNA
CUGGUGUCCACUGCUUUAGTT
1612

(967C)

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 988L21 antisense siNA
CUCCUGGUGUCCACUGCUUTT
1613

(970C)

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1211L21 antisense siNA
CAGUGUUCAAGGUACCCUUTT
1614

(1193C)

391
CCCAGUUUGUAAAGGACCUGCUC
1285

IL13: 393U21 sense siNA stab04
B cAGuuuGuAAAGGAccuGcTT B
1615

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 799U21 sense siNA stab04
B cuucAcAcAcAGGcAAcuGTT B
1616

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 834U21 sense siNA stab04
B AGGcAcAcuucuucuuGGuTT B
1617

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 913U21 sense siNA stab04
B GAcuGuGGcuGcuAGcAcuTT B
1618

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 965U21 sense siNA stab04
B cAcuAAAGcAGuGGAcAccTT B
1619

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 967U21 sense siNA stab04
B cuAAAGcAGuGGAcAccAGTT B
1620

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 970U21 sense siNA stab04
B AAGcAGuGGAcAccAGGAGTT B
1621

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1193U21 sense siNA stab04
B AAGGGuAccuuGAAcAcuGTT B
1622

391
CCCAGUUUGUAAAGGACCUGCUC
1285

IL13: 411L21 antisense siNA
GcAGGuccuuuAcAAAcuGTsT
1623

(393C) stab05

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 817L21 antisense siNA
cAGuuGccuGuGuGuGAAGTsT
1624

(799C) stab05

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 852L21 antisense siNA
AccAAGAAGAAGuGuGccuTsT
1625

(834C) stab05

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 931L21 antisense siNA
AGuGcuAGcAGccAcAGucTsT
1626

(913C) stab05

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 983L21 antisense siNA
GGuGuccAcuGcuuuAGuGTsT
1627

(965C) stab05

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 985L21 antisense siNA
cuGGuGuccAcuGcuuuAGTsT
1628

(967C) stab05

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 988L21 antisense siNA
cuccuGGuGuccAcuGcuuTsT
1629

(970C) stab05

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1211L21 antisense siNA
cAGuGuucAAGGuAcccuuTsT
1630

(1193C) stab05

864
UAUUGUGUGUUAUUUAAAUGAGU
1293
33355
IL13: 864U21 sense siNA stab07
B uuGuGuGuuAuuuAAAuGATT B
1631

865
AUUGUGUGUUAUUUAAAUGAGUG
1294
33356
IL13: 865U21 sense siNA stab07
B uGuGuGuuAuuuAAAuGAGTT B
1632

866
UUGUGUGUUAUUUAAAUGAGUGU
1295
33357
IL13: 866U21 sense siNA stab07
B GuGuGuuAuuuAAAuGAGuTT B
1633

863
UUAUUGUGUGUUAUUUAAAUGAG
1296
33358
IL13: 863U21 sense siNA stab07
B AuuGuGuGuuAuuuAAAuGTT B
1634

200
UGCAAUGGCAGCAUGGUAUGGAG
1297
33359
IL13: 200U21 sense siNA stab07
B cAAuGGcAGcAuGGuAuGGTT B
1635

201
GCAAUGGCAGCAUGGUAUGGAGC
1298
33360
IL13: 201U21 sense siNA stab07
B AAuGGcAGcAuGGuAuGGATT B
1636

202
CAAUGGCAGCAUGGUAUGGAGCA
1299
33361
IL13: 202U21 sense siNA stab07
B AuGGcAGcAuGGuAuGGAGTT B
1637

860
UUAUUAUUGUGUGUUAUUUAAAU
1300
33362
IL13: 860U21 sense siNA stab07
B AuuAuuGuGuGuuAuuuAATT B
1638

861
UAUUAUUGUGUGUUAUUUAAAUG
1301
33363
IL13: 861U21 sense siNA stab07
B uuAuuGuGuGuuAuuuAAATT B
1639

862
AUUAUUGUGUGUUAUUUAAAUGA
1302
33364
IL13: 862U21 sense siNA stab07
B uAuuGuGuGuuAuuuAAAuTT B
1640

391
CCCAGUUUGUAAAGGACCUGCUC
1285

IL13: 393U21 sense siNA stab07
B cAGuuuGuAAAGGAccuGcTT B
1641

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 799U21 sense siNA stab07
B cuucAcAcAcAGGcAAcuGTT B
1642

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 834U21 sense siNA stab07
B AGGcAcAcuucuucuuGGuTT B
1643

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 913U21 sense siNA stab07
B GAcuGuGGcuGcuAGcAcuTT B
1644

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 965U21 sense siNA stab07
B cAcuAAAGcAGuGGAcAccTT B
1645

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 967U21 sense siNA stab07
B cuAAAGcAGuGGAcAccAGTT B
1646

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 970U21 sense siNA stab07
B AAGcAGuGGAcAccAGGAGTT B
1647

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1193U21 sense siNA stab07
B AAGGGuAccuuGAAcAcuGTT B
1648

391
CCCAGUUUGUAAAGGACCUGCUC
1285

IL13: 411L21 antisense siNA

GcAGGuccuuuAcAAAcuGTsT
1649

(393C) stab11

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 817L21 antisense siNA
cAGuuGccuGuGuGuGAAGTsT
1650

(799C) stab11

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 852L21 antisense siNA

AccAAGAAGAAGuGuGccuTsT
1651

(834C) stab11

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 931L21 antisense siNA

AGuGcuAGcAGccAcAGucTsT
1652

(913C) stab11

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 983L21 antisense siNA

GGuGuccAcuGcuuuAGuGTsT
1653

(965C) stab11

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 985L21 antisense siNA
cuGGuGuccAcuGcuuuAGTsT
1654

(967C) stab11

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 988L21 antisense siNA
cuccuGGuGuccAcuGcuuTsT
1655

(970C) stab11

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1211L21 antisense siNA
cAGuGuucAAGGuAcccuuTsT
1656

(1193C) stab11

391
CCCAGUUUGUAAAGGACCUGCUC
1285

IL13: 393U21 sense siNA stab18
B cAGuuuGuAAAGGAccuGcTT B
1657

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 799U21 sense siNA stab18
B cuucAcAcAcAGGcAAcuGTT B
1658

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 834U21 sense siNA stab18
B AGGcAcAcuucuucuuGGuTT B
1659

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 913U21 sense siNA stab18
B GAcuGuGGcuGcuAGcAcuTT B
1660

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 965U21 sense siNA stab18
B cAcuAAAGcAGuGGAcAccTT B
1661

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 967U21 sense siNA stab18
B cuAAAGcAGuGGAcAccAGTT B
1662

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 970U21 sense siNA stab18
B AAGcAGuGGAcAccAGGAGTT B
1663

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1193U21 sense siNA stab18
B AAGGGuAccuuGAAcAcuGTT B
1664

864
UAUUGUGUGUUAUUUAAAUGAGU
1293
33375
IL13: 882L21 antisense siNA
ucAuuuAAAuAAcAcAcAATsT
1665

(864C) stab08

865
AUUGUGUGUUAUUUAAAUGAGUG
1294
33376
IL13: 883L21 antisense siNA
cucAuuuAAAuAAcAcAcATsT
1666

(865C) stab08

866
UUGUGUGUUAUUUAAAUGAGUGU
1295
33377
IL13: 884L21 antisense siNA

AcucAuuuAAAuAAcAcAcTsT
1667

(866C) stab08

863
UUAUUGUGUGUUAUUUAAAUGAG
1296
33378
IL13: 881L21 antisense siNA
cAuuuAAAuAAcAcAcAAuTsT
1668

(863C) stab08

200
UGCAAUGGCAGCAUGGUAUGGAG
1297
33379
IL13: 218L21 antisense siNA
ccAuAccAuGcuGccAuuGTsT
1669

(200C) stab08

201
GCAAUGGCAGCAUGGUAUGGAGC
1298
33380
IL13: 219L21 antisense siNA
uccAuAccAuGcuGccAuuTsT
1670

(201C) stab08

202
CAAUGGCAGCAUGGUAUGGAGCA
1299
33381
IL13: 220L21 antisense siNA
cuccAuAccAuGcuGccAuTsT
1671

(202C) stab08

860
UUAUUAUUGUGUGUUAUUUAAAU
1300
33382
IL13: 878L21 antisense siNA
uuAAAuAAcAcAcAAuAAuTsT
1672

(860C) stab08

861
UAUUAUUGUGUGUUAUUUAAAUG
1301
33383
IL13: 879L21 antisense siNA
uuuAAAuAAcAcAcAAuAATsT
1673

(861C) stab08

862
AUUAUUGUGUGUUAUUUAAAUGA
1302
33384
IL13: 880L21 antisense siNA

AuuuAAAuAAcAcAcAAuATsT
1674

(862C) stab08

391
CCCAGUUUGUAAAGGACCUGCUC
1285

IL13: 411L21 antisense siNA

GcAGGuccuuuAcAAAcuGTsT
1675

(393C) stab08

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 817L21 antisense siNA
cAGuuGccuGuGuGuGAAGTsT
1676

(799C) stab08

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 852L21 antisense siNA

AccAAGAAGAAGuGuGccuTsT
1677

(834C) stab08

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 931L21 antisense siNA

AGuGcuAGcAGccAcAGucTsT
1678

(913C) stab08

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 983L21 antisense siNA

GGuGuccAcuGcuuuAGuGTsT
1679

(965C) stab08

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 985L21 antisense siNA
cuGGuGuccAcuGcuuuAGTsT
1680

(967C) stab08

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 988L21 antisense siNA
cuccuGGuGuccAcuGcuuTsT
1681

(970C) stab08

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1211L21 antisense siNA
cAGuGuucAAGGuAcccuuTsT
1682

(1193C) stab08

391
CCCAGUUUGUAAAGGACCUGCUC
1285
36890
IL13: 393U21 sense siNA stab09
B CAGUUUGUAAAGGACCUGCTT B
1683

797
CACUUCACACACAGGCAACUGAG
1286
36891
IL13: 799U21 sense siNA stab09
B CUUCACACACAGGCAACUGTT B
1684

832
UCAGGCACACUUCUUCUUGGUCU
1287
36892
IL13: 834U21 sense siNA stab09
B AGGCACACUUCUUCUUGGUTT B
1685

911
AAGACUGUGGCUGCUAGCACUUG
1288
36893
IL13: 913U21 sense siNA stab09
B GACUGUGGCUGCUAGCACUTT B
1686

963
AGCACUAAAGCAGUGGACACCAG
1289
36894
IL13: 965U21 sense siNA stab09
B CACUAAAGCAGUGGACACCTT B
1687

965
CACUAAAGCAGUGGACACCAGGA
1290
36895
IL13: 967U21 sense siNA stab09
B CUAAAGCAGUGGACACCAGTT B
1688

968
UAAAGCAGUGGACACCAGGAGUC
1291
36896
IL13: 970U21 sense siNA stab09
B AAGCAGUGGACACCAGGAGTT B
1689

1191
AGAAGGGUACCUUGAACACUGGG
1292
36897
IL13: 1193U21 sense siNA stab09
B AAGGGUACCUUGAACACUGTT B
1690

391
CCCAGUUUGUAAAGGACCUGCUC
1285

IL13: 411L21 antisense siNA
GCAGGUCCUUUACAAACUGTsT
1691

(393C) stab10

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 817L21 antisense siNA
CAGUUGCCUGUGUGUGAAGTsT
1692

(799C) stab10

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 852L21 antisense siNA
ACCAAGAAGAAGUGUGCCUTsT
1693

(834C) stab10

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 931L21 antisense siNA
AGUGCUAGCAGCCACAGUCTsT
1694

(913C) stab10

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 983L21 antisense siNA
GGUGUCCACUGCUUUAGUGTsT
1695

(965C) stab10

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 985L21 antisense siNA
CUGGUGUCCACUGCUUUAGTsT
1696

(967C) stab10

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 988L21 antisense siNA
CUCCUGGUGUCCACUGCUUTsT
1697

(970C) stab10

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1211L21 antisense siNA
CAGUGUUCAAGGUACCCUUTsT
1698

(1193C) stab10

391
CCCAGUUUGUAAAGGACCUGCUC
1285

IL13: 411L21 antisense siNA
GcAGGuccuuuAcAAAcuGTT B
1699

(393C) stab19

797
CACUUCACACACAGGCAACUGAG
1286

IL13: 817L21 antisense siNA
cAGuuGccuGuGuGuGAAGTT B
1700

(799C) stab19

832
UCAGGCACACUUCUUCUUGGUCU
1287

IL13: 852L21 antisense siNA
AccAAGAAGAAGuGuGccuTT B
1701

(834C) stab19

911
AAGACUGUGGCUGCUAGCACUUG
1288

IL13: 931L21 antisense siNA
AGuGcuAGcAGccAcAGucTT B
1702

(913C) stab19

963
AGCACUAAAGCAGUGGACACCAG
1289

IL13: 983L21 antisense siNA
GGuGuccAcuGcuuuAGuGTT B
1703

(965C) stab19

965
CACUAAAGCAGUGGACACCAGGA
1290

IL13: 985L21 antisense siNA
cuGGuGuccAcuGcuuuAGTT B
1704

(967C) stab19

968
UAAAGCAGUGGACACCAGGAGUC
1291

IL13: 988L21 antisense siNA
cuccuGGuGuccAcuGcuuTT B
1705

(970C) stab19

1191
AGAAGGGUACCUUGAACACUGGG
1292

IL13: 1211L21 antisense siNA
cAGuGuucAAGGuAcccuuTT B
1706

(1193C) stab19

391
CCCAGUUUGUAAAGGACCUGCUC
1285
36898
IL13: 411L21 antisense siNA
GCAGGUCCUUUACAAACUGTT B
1707

(393C) stab22

797
CACUUCACACACAGGCAACUGAG
1286
36899
IL13: 817L21 antisense siNA
CAGUUGCCUGUGUGUGAAGTT B
1708

(799C) stab22

832
UCAGGCACACUUCUUCUUGGUCU
1287
36900
IL13: 852L21 antisense siNA
ACCAAGAAGAAGUGUGCCUTT B
1709

(834C) stab22

911
AAGACUGUGGCUGCUAGCACUUG
1288
36901
IL13: 931L21 antisense siNA
AGUGCUAGCAGCCACAGUCTT B
1710

(913C) stab22

963
AGCACUAAAGCAGUGGACACCAG
1289
36902
IL13: 983L21 antisense siNA
GGUGUCCACUGCUUUAGUGTT B
1711

(965C) stab22

965
CACUAAAGCAGUGGACACCAGGA
1290
36903
IL13: 985L21 antisense siNA
CUGGUGUCCACUGCUUUAGTT B
1712

(967C) stab22

968
UAAAGCAGUGGACACCAGGAGUC
1291
36904
IL13: 988L21 antisense siNA
CUCCUGGUGUCCACUGCUUTT B
1713

(970C) stab22

1191
AGAAGGGUACCUUGAACACUGGG
1292
36905
IL13: 1211L21 antisense siNA
CAGUGUUCAAGGUACCCUUTT B
1714

(1193C) stab22

IL13R

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 410U21 sense siNA
GGUGAUCCUGAGUCUGCUGTT
1715

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 659U21 sense siNA
GUCAAGGAUAAUGCAGGAATT
1716

871
CGUCCAAGAGGCUAAAUGUGAGA
1305

IL13RA1: 873U21 sense siNA
UCCAAGAGGCUAAAUGUGATT
1717

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1: 1278U21 sense siNA
AAACCGACUCUGUAGUGCUTT
1718

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1310U21 sense siNA
AAGAAAGCCUCUCAGUGAUTT
1719

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1426U21 sense siNA
UGCACCAUUUAAAAACAGGTT
1720

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2188U21 sense siNA
GCAUUUUCCUCUGCUUUGATT
1721

2270
CCAAGACCUUUCAAAGCCAUUUU
1310

IL13RA1: 2272U21 sense siNA
AAGACCUUUCAAAGCCAUUTT
1722

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 428L21 antisense siNA
CAGCAGACUCAGGAUCACCTT
1723

(410C)

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 677L21 antisense siNA
UUCCUGCAUUAUCCUUGAGTT
1724

(659C)

871
CGUCCAAGAGGCUAAAUGUGAGA
1305

IL13RA1: 891L21 antisense siNA
UCACAUUUAGCCUCUUGGATT
1725

(873C)

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1: 1296L21 antisense siNA
AGCACUACAGAGUCGGUUUTT
1726

(1278C)

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1328L21 antisense siNA
AUCACUGAGAGGCUUUCUUTT
1727

(1310C)

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1444L21 antisense siNA
CCUGUUUUUAAAUGGUGCATT
1728

(1426C)

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2206L21 antisense siNA
UCAAAGCAGAGGAAAAUGCTT
1729

(2188C)

2270
CCAAGACCUUUCAAAGCCAUUUU
1310

IL13RA1: 2290L21 antisense siNA
AAUGGCUUUGAAAGGUCUUTT
1730

(2272C)

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 410U21 sense siNA
B GGuGAuccuGAGucuGcuGTT B
1731

stab04

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 659U21 sense siNA
B GucAAGGAuAAuGcAGGAATT B
1732

stab04

871
CGUCCAAGAGGCUAAAUGUGAGA
1305

IL13RA1: 873U21 sense siNA
B uccAAGAGGcuAAAuGuGATT B
1733

stab04

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1: 1278U21 sense siNA
B AAAccGAcucuGuAGuGcuTT B
1734

stab04

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1310U21 sense siNA
B AAGAAAGccucucAGuGAuTT B
1735

stab04

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1426U2l sense siNA
B uGcAccAuuuAAAAAcAGGTT B
1736

stab04

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2188U21 sense siNA
B GcAuuuuccucuGcuuuGATT B
1737

stab04

2270
CCAAGACCUUUCAAAGCCAUUUU
1310

IL13RA1: 2272U21 sense siNA
B AAGAccuuucAAAGccAuuTT B
1738

stab04

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 428L21 antisense siNA
cAGcAGAcucAGGAucAccTsT
1739

(410C) stab05

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 677L21 antisense siNA
uuccuGcAuuAuccuuGAcTsT
1740

(659C) stab05

871
CGUCCAAGAGGCUAAAUGUGAGA
1305

IL13RA1: 891L21 antisense siNA
ucAcAuuuAGccucuuGGATsT
1741

(873C) stab05

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1:1296L21 antisense siNA
AGcAcuAcAGAGucGGuuuTsT
1742

(1278C) stab05

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1328L21 antisense siNA
AucAcuGAGAGGcuuucuuTsT
1743

(1310C) stab05

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1444L21 antisense siNA
ccuGuuuuuAAAuGGuGcATsT
1744

(1426C) stab05

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2206L21 antisense siNA
ucAAAGcAGAGGAAAAuGcTsT
1745

(2188C) stab05

2270
CCAAGACCUUUCAAAGCCAUUUU
1310

IL13RA1: 2290L21 antisense siNA
AAuGGcuuuGAAAGGucuuTsT
1746

(2272C) stab05

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 410U21 sense siNA
B GGuGAuccuGAGucuGcuGTT B
1747

stab07

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 659U21 sense siNA
B GucAAGGAuAAuGcAGGAATT B
1748

stab07

871
CGUCCAAGAGGCUAAAUGUGAGA
1305

IL13RA1: 873U21 sense siNA
B uccAAGAGGcuAAAuGuGATT B
1749

stab07

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1: 1278U21 sense siNA
B AAAccGAcucuGuAGuGcuTT B
1750

stab07

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1310U21 sense siNA
B AAGAAAGccucucAGuGAuTT B
1751

stab07

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1426U21 sense siNA
B uGcAccAuuuAAAAAcAGGTT B
1752

stab07

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2188U21 sense siNA
B GcAuuuuccucuGcuuuGATT B
1753

stab07

2270
CCAAGACCUUUCAAAGCCAUUUU
1310

IL13RA1: 2272U21 sense siNA
B AAGAccuuucAAAGccAuuTT B
1754

stab07

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 428L21 antisense siNA
cAGcAGAcucAGGAucAccTsT
1755

(410C) stab11

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 677L21 antisense siNA
uuccuGcAuuAuccuuGAcTsT
1756

(659C) stab11

871
CGUCCAAGAGGCUAAAUGUGAGA
1305

IL13RA1: 891L21 antisense siNA
ucAcAuuuAGccucuuGGATsT
1757

(873C) stab11

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1: 1296L21 antisense siNA

AGcAcuAcAGAGucGGuuuTsT
1758

(1278C) stab11

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1328L21 antisense siNA

AucAcuGAGAGGcuuucuuTsT
1759

(1310C) stab11

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1444L21 antisense siNA
ccuGuuuuuAAAuGGuGcATsT
1760

(1426C) stab11

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2206L21 antisense siNA
ucAAAGcAGAGGAAAAuGcTsT
1761

(2188C) stab11

2270
CCAAGACGUUUCAAAGCCAUUUU
1310

IL13RA1: 2290L21 antisense siNA
AAuGGcuuuGAAAGGucuuTsT
1762

(2272C) stab11

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 410U21 sense siNA
B GGuGAuccuGAGucuGcuGTT B
1763

stab18

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 659U21 sense siNA
B GucAAGGAuAAuGcAGGAATT B
1764

stab18

871
CGUCCAAGAGGCUAAAUGUGAGA
1305

IL13RA1: 873U21 sense siNA
B uccAAGAGGcuAAAuGuGATT B
1765

stab18

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1: 1278U21 sense siNA
B AAAccGAcucuGuAGuGcuTT B
1766

stab18

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1310U21 sense siNA
B AAGAAAGccucucAGuGAuTT B
1767

stab18

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1426U21 sense siNA
B uGcAccAuuuAAAAAcAGGTT B
1768

stab18

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2188U21 sense siNA
B GcAuuuuccucuGcuuuGATT B
1769

stab18

2270
CCAAGACCUUUCAAAGCCAUUUU
1310

IL13RA1: 2272U21 sense siNA
B AAGAccuuucAAAGccAuuTT B
1770

stab18

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 428L21 antisense siNA
cAGcAGAcucAGGAucAccTsT
1771

(410C) stab08

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 677L21 antisense siNA
uuccuGcAuuAuccuuGAcTsT
1772

(659C) stab08

871
CGUCCAAGAGGCUAAAUGUGAGA
1305

IL13RA1: 891L21 antisense siNA
ucAcAuuuAGccucuuGGATsT
1773

(873C) stab08

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1: 1296L21 antisense siNA

AGcAcuAcAGAGucGGuuuTsT
1774

(1278C) stab08

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1328L21 antisense siNA

AucAcuGAGAGGcuuucuuTsT
1775

(1310C) stab08

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1444L21 antisense siNA
ccuGuuuuuAAAuGGuGcATsT
1776

(1426C) stab08

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2206L21 antisense siNA
ucAAAGcAGAGGAAAAuGcTsT
1777

(2188C) stab08

2270
CCAAGACCUUUCAAAGCCAUUUU
1310

IL13RA1: 2290L21 antisense siNA

AAuGGcuuuGAAAGGucuuTsT
1778

(2272C) stab08

408
AAGGUGAUCCUGAGUCUGCUGUG
1303
36906
IL13RA1: 410U21 sense siNA
B GGUGAUCCUGAGUCUGCUGTT B
1779

stab09

657
UGGUCAAGGAUAAUGCAGGAAAA
1304
36907
IL13RA1: 659U21 sense siNA
B GUCAAGGAUAAUGCAGGAATT B
1780

stab09

871
CGUCCAAGAGGCUAAAUGUGAGA
1305
36908
IL13RA1: 873U21 sense siNA
B UCCAAGAGGCUAAAUGUGATT B
1781

stab09

1276
GGAAACCGACUCUGUAGUGCUGA
1306
36909
IL13RA1: 1278U21 sense siNA
B AAACCGACUCUGUAGUGCUTT B
1782

stab09

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307
36910
IL13RA1: 1310U21 sense siNA
B AAGAAAGCCUCUCAGUGAUTT B
1783

stab09

1424
ACUGCACCAUUUAAAAACAGGCA
1308
36911
IL13RA1: 1426U21 sense siNA
B UGCACCAUUUAAAAACAGGTT B
1784

stab09

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309
36912
IL13RA1: 2188U21 sense siNA
B GCAUUUUCCUCUGCUUUGATT B
1785

stab09

2270
CCAAGACCUUUCAAAGCCAUUUU
1310
36913
IL13RA1: 2272U21 sense siNA
B AAGACCUUUCAAAGCCAUUTT B
1786

stab09

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 428L21 antisense siNA
CAGCAGACUCAGGAUCACCTsT
1787

(410C) stab10

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 677L21 antisense siNA
UUCCUGCAUUAUCCUUGACTsT
1788

(659C) stab10

871
CGUCCAAGAGGCUAAAUGUGAGA
1305

IL13RA1: 891L21 antisense siNA
UCACAUUUAGCCUCUUGGATsT
1789

(873C) stab10

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1: 1296L21 antisense siNA
AGCACUACAGAGUCGGUUUTsT
1790

(1278C) stab10

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1328L21 antisense siNA
AUCACUGAGAGGCUUUCUUTsT
1791

(1310C) stab10

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1444L21 antisense siNA
CCUGUUUUUAAAUGGUGCATsT
1792

(1426C) stab10

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2206L21 antisense siNA
UCAAAGCAGAGGAAAAUGCTsT
1793

(2188C) stab10

2270
CCAAGACCUUUCAAAGCCAUUUU
1310

IL13RA1: 2290L21 antisense siNA
AAUGGCUUUGAAAGGUCUUTsT
1794

(2272C) stab10

408
AAGGUGAUCCUGAGUCUGCUGUG
1303

IL13RA1: 428L21 antisense siNA
CAGcAGAcucAGGAucAccTT B
1795

(410C) stab19

657
UGGUCAAGGAUAAUGCAGGAAAA
1304

IL13RA1: 677L21 antisense siNA
uuccuGcAuuAuccuuGAcTT B
1796

(659C) stab19

871
CGUCCAAGAGGCUAAAUGLiGAGA
1305

IL13RA1: 891L21 antisense siNA
ucAcAuuuAGccucuuGGATT B
1797

(873C) stab19

1276
GGAAACCGACUCUGUAGUGCUGA
1306

IL13RA1: 1296L21 antisense siNA
AGcAcuAcAGAGucGGuuuTT B
1798

(1278C) stab19

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307

IL13RA1: 1328L21 antisense siNA
AucAcuGAGAGGcuuucuuTT B
1799

(1310C) stab19

1424
ACUGCACCAUUUAAAAACAGGCA
1308

IL13RA1: 1444L21 antisense siNA
ccuGuuuuuAAAuGGuGcATT B
1800

(1426C) stab19

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309

IL13RA1: 2206L21 antisense siNA
ucAAAGcAGAGGAAAAuGcTT B
1801

(2188C) stab19

2270
CCAAGACCUUUCAAAGCCAUUUU
1310

IL13RA1: 2290L21 antisense siNA
AAuGGcuuuGAAAGGucuuTT B
1802

(2272C) stab19

408
AAGGUGAUCCUGAGUCUGCUGUG
1303
36914
IL13RA1: 428L21 antisense siNA
CAGCAGACUCAGGAUCACCTT B
1803

(410C) stab22

657
UGGUCAAGGAUAAUGCAGGAAAA
1304
36915
IL13RA1: 677L21 antisense siNA
UUCCUGCAUUAUCCUUGACTT B
1804

(659C) stab22

871
CGUCCAAGAGGCUAAAUGUGAGA
1305
36916
IL13RA1: 891L21 antisense siNA
UCACAUUUAGCCUCUUGGATT B
1805

(873C) stab22

1276
GGAAACCGACUCUGUAGUGCUGA
1306
36917
IL13RA1: 1296L21 antisense siNA
AGCACUACAGAGUCGGUUUTT B
1806

(1278C) stab22

1308
UGAAGAAAGCCUCUCAGUGAUGG
1307
36918
IL13RA1: 1328L21 antisense siNA
AUCACUGAGAGGCUUUCUUTT B
1807

(1310C) stab22

1424
ACUGCACCAUUUAAAAACAGGCA
1308
36919
IL13RA1: 1444L21 antisense siNA
CCUGUUUUUAAAUGGUGCATT B
1808

(1426C) stab22

2186
CAGCAUUUUCCUCUGCUUUGAAA
1309
36920
IL13RA1: 2206L21 antisense siNA
UCAAAGCAGAGGAAAAUGCTTB
1809

(2188C) stab22

2270
CCAAGACCUUUCAAAGCCAUUUU
1310
36921
IL13RA1: 2290L21 antisense siNA
AAUGGCUUUGAAAGGUCUUTT B
1810

(2272C) stab22

Non-Human IL and ILR

222
UGCAACGGCAGCAUGGUAUGGAG
1811
33365
mIL13: 222U21 sense siNA stab07
B cAAcGGcAGcAuGGuAuGGTT B
1981

223
GCAACGGCAGCAUGGUAUGGAGU
1812
33366
mIL13: 223U21 sense siNA stab07
B AAcGGcAGcAuGGuAuGGATT B
1982

224
CAACGGCAGCAUGGUAUGGAGUG
1813
33367
mIL13: 224U21 sense siNA stab07
B AcGGcAGcAuGGuAuGGAGTT B
1983

780
UUAUGGUUGUGUGUUAUUUAAAU
1814
33368
mIL13: 780U21 sense siNA stab07
B AuGGuuGuGuGuuAuuuAATT B
1984

781
UAUGGUUGUGUGUUAUUUAAAUG
1815
33369
mIL13: 781U21 sense siNA stab07
B uGGuuGuGuGuuAuuuAAATT B
1985

782
AUGGUUGUGUGUUAUUUAAAUGA
1816
33370
mIL13: 782U21 sense siNA stab07
B GGuuGuGuGuuAuuuAAAuTT B
1986

783
UGGUUGUGUGUUAUUUAAAUGAG
1817
33371
mIL13: 783U21 sense siNA stab07
B GuuGuGuGuuAuuuAAAuGTT B
1987

906
CAUAACUCUGCUACCUCACUGUA
1818
33372
mIL13: 906U21 sense siNA stab07
B uAAcucuGcuAccucAcuGTT B
1988

1057
AAUAGCUUAGCAAAGAGUUAAUA
1819
33373
mIL13: 1057U21 sense siNA
B uAGcuuAGcAAAGAGuuAATT B
1989

stab07

1059
UAGCUUAGCAAAGAGUUAAUAAU
1820
33374
mIL13: 1059U21 sense siNA
B GcuuAGcAAAGAGuuAAuATT B
1990

stab07

222
UGCAACGGCAGCAUGGUAUGGAG
1811
33385
mIL13: 240L21 antisense siNA
ccAuAccAuGcuGccGuuGTsT
1991

(222C) stab08

223
GCAACGGCAGCAUGGUAUGGAGU
1812
33386
mIL13: 241L21 antisense siNA
uccAuAccAuGcuGccGuuTsT
1992

(223C) stab08

224
CAACGGCAGCAUGGUAUGGAGUG
1813
33387
mIL13: 242L21 antisense siNA
cuccAuAccAuGcuGccGuTsT
1993

(224C) stab08

780
UUAUGGUUGUGUGUUAUUUAAAU
1814
33388
mIL13: 798L21 antisense siNA
uuAAAuAAcAcAcAAccAuTsT
1994

(780C) stab08

781
UAUGGUUGUGUGUUAUUUAAAUG
1815
33389
mIL13: 799L21 antisense siNA
uuuAAAuAAcAcAcAAccATsT
1995

(781C) stab08

782
AUGGUUGUGUGUUAUUUAAAUGA
1816
33390
mIL13: 800L21 antisense siNA

AuuuAAAuAAcAcAcAAccTsT
1996

(782C) stab08

783
UGGUUGUGUGUUAUUUAAAUGAG
1817
33391
mIL13: 801L21 antisense siNA
cAuuuAAAuAAcAcAcAAcTsT
1997

(783C) stab08

906
CAUAACUCUGCUACCUCACUGUA
1818
33392
mIL13: 924L21 antisense siNA
cAGuGAGGuAGcAGAGuuATsT
1998

(906C) stab08

1057
AAUAGCUUAGCAAAGAGUUAAUA
1819
33393
mIL13: 1075L21 antisense siNA
uuAAcucuuuGcuAAGcuATsT
1999

(1057C) stab08

1059
UAGCUUAGCAAAGAGUUAAUAAU
1820
33394
mIL13: 1077L21 antisense siNA
uAuuAAcucuuuGcuAAGcTsT
2000

(1059C) stab08

11
CUGGGUGACUGCAGUCCUGGCUC
1821
38093
rIL13: 11U21 sense siNA stab07
B GGGuGAcuGcAGuccuGGcTT B
2001

14
GGUGACUGCAGUCCUGGCUCUCG
1822
38094
rIL13: 14U21 sense siNA stab07
B uGAcuGcAGuccuGGcucuTT B
2002

15
GUGACUGCAGUCCUGGCUCUCGC
1823
38095
rIL13: 15U21 sense siNA stab07
B GAcuGcAGuccuGGcucucTT B
2003

16
UGACUGCAGUCCUGGCUCUCGCU
1824
38096
rIL13: 16U21 sense siNA stab07
B AcuGcAGuccuGGcucucGTT B
2004

17
GACUGCAGUCCUGGCUCUCGCUU
1825
38097
rIL13: 17U21 sense siNA stab07
B cuGcAGuccuGGcucucGcTT B
2005

99
CUCAGGGAGCUUAUCGAGGAGCU
1826
38098
rIL13: 99U21 sense siNA stab07
B cAGGGAGcuuAucGAGGAGTT B
2006

113
CGAGGAGCUGAGCAACAUCACAC
1827
38099
rIL13: 113U21 sense siNA stab07
B AGGAGcuGAGcAAcAucAcTT B
2007

114
GAGGAGCUGAGCAACAUCACACA
1828
38100
rIL13: 114U21 sense siNA stab07
B GGAGcuGAGcAAcAucAcATT B
2008

115
AGGAGCUGAGCAACAUCACACAA
1829
38101
rIL13: 115U21 sense siNA stab07
B GAGcuGAGcAAcAucAcAcTT B
2009

116
GGAGCUGAGCAACAUCACACAAG
1830
38102
rIL13: 116U21 sense siNA stab07
B AGcuGAGcAAcAucAcAcATT B
2010

117
GAGCUGAGCAACAUCACACAAGA
1831
38103
rIL13: 117U21 sense siNA stab07
B GcuGAGcAAcAucAcAcAATT B
2011

120
CUGAGCAACAUCACACAAGACCA
1832
38104
rIL13: 120U21 sense siNA stab07
B GAGcAAcAucAcAcAAGAcTT B
2012

121
UGAGCAACAUCACACAAGACCAG
1833
38105
rIL13: 121U21 sense siNA stab07
B AGcAAcAucAcAcAAGAccTT B
2013

122
GAGCAACAUCACACAAGACCAGA
1834
38106
rIL13: 122U21 sense siNA stab07
B GcAAcAucAcAcAAGAccATT B
2014

123
AGCAACAUCACACAAGACCAGAA
1835
38107
rIL13: 123U21 sense siNA stab07
B cAAcAucAcAcAAGAccAGTT B
2015

124
GCAACAUCACACAAGACCAGAAG
1836
38108
rIL13: 124U21 sense siNA stab07
B AAcAucAcAcAAGAccAGATT B
2016

141
CAGAAGACUUCCCUGUGCAACAG
1837
38109
rIL13: 141U21 sense siNA stab07
B GAAGAcuucccuGuGcAAcTT B
2017

159
AACAGCAGCAUGGUAUGGAGCGU
1838
38110
rIL13: 159U21 sense siNA stab07
B cAGcAGcAuGGuAuGGAGcTT B
2018

188
GACAGCUGGCGGGUUCUGUGCAG
1839
38111
rIL13: 188U21 sense siNA stab07
B cAGcuGGcGGGuucuGuGcTT B
2019

217
AAUCCCUGACCAACAUCUCCAGU
1840
38112
rIL13: 217U21 sense siNA stab07
B ucccuGAccAAcAucuccATT B
2020

237
AGUUGCAAUGCCAUCCACAGGAC
1841
38113
rIL13: 237U21 sense siNA stab07
B uuGcAAuGccAuccAcAGGTT B
2021

252
CACAGGACCCAGAGGAUAUUGAA
1842
38114
rIL13: 252U21 sense siNA stab07
B cAGGAcccAGAGGAuAuuGTT B
2022

319
CAGAUACCAAAAUCGAAGUAGCC
1843
38115
rIL13: 319U21 sense siNA stab07
B GAuAccAAAAucGAAGuAGTT B
2023

320
AGAUACCAAAAUCGAAGUAGCCC
1844
38116
rIL13: 320U21 sense siNA stab07
B AuAccAAAAucGAAGuAGcTT B
2024

321
GAUACCAAAAUCGAAGUAGCCCA
1845
38117
rIL13: 321U21 sense siNA stab07
B uAccAAAAucGAAGuAGccTT B
2025

322
AUACCAAAAUCGAAGUAGCCCAG
1846
38118
rfLI3: 322U21 sense siNA stab07
B AccAAAAucGAAGuAGcccTT B
2026

323
UACCAAAAUCGAAGUAGCCCAGU
1847
38119
rIL13: 323U21 sense siNA stab07
B ccAAAAucGAAGuAGcccATT B
2027

360
CUCAAUUACUCCAAGCAACUUUU
1848
38120
rIL13: 360U21 sense siNA stab07
B cAAuuAcuccAAGcAAcuuTT B
2028

361
UCAAUUACUCCAAGCAACUUUUC
1849
38121
rIL13: 361U21 sense siNA stab07
B AAuuAcuccAAGcAAcuuuTT B
2029

362
CAAUUACUCCAAGCAACUUUUCC
1850
38122
rIL13: 362U21 sense siNA stab07
B AuuAcuccAAGcAAcuuuuTT B
2030

375
CAACUUUUCCGCUAUGGCCACUG
1851
38123
rIL13: 375U21 sense siNA stab07
B AcuuuuccGcuAuGGccAcTT B
2031

420
CUCAGCUGUGGACCUCAGUUGUG
1852
38124
rIL13: 420U21 sense siNA stab07
B cAGcuGuGGAccucAGuuGTT B
2032

11
CUGGGUGACUGCAGUCCUGGCUC
1821
38125
rIL13: 29L21 antisense siNA

GCCAGGAcuGcAGucAcccTT
2033

(11C) stab26

14
GGUGACUGCAGUCCUGGCUCUCG
1822
38126
rIL13: 32L21 antisense siNA

AGAGccAGGAcuGcAGucATT
2034

(14C) stab26

15
GUGACUGCAGUCCUGGCUCUCGC
1823
38127
rIL13: 33L21 antisense siNA

GAGAGccAGGAcuGcAGucTT
2035

(15C) stab26

16
UGACUGCAGUCCUGGCUCUCGCU
1824
38128
rIL13: 34L21 antisense siNA
CGAGAGccAGGAcuGcAGuTT
2036

(16C) stab26

17
GACUGCAGUCCUGGCUCUCGCUU
1825
38129
rIL13: 35L21 antisense siNA

GCGAGAGccAGGAcuGcAGTT
2037

(17C) stab26

99
CUCAGGGAGCUUAUCGAGGAGCU
1826
38130
rIL13: 117L21 antisense siNA
CUCcucGAuAAGcucccuGTT
2038

(99C) stab26

113
CGAGC3AGCUGAGCAACAUCACAC
1827
38131
rIL13: 131L21 antisense siNA

GUGAuGuuGcucAGcuccuTT
2039

(113C) stab26

114
GAGGAGCUGAGCAACAUCACACA
1828
38132
rIL13: 132L21 antisense siNA
UGUGAUGuuGcucAGcuccTT
2040

(114C) stab26

115
AGGAGCUGAGCAACAUCACACAA
1829
38133
rIL13: 133L21 antisense siNA

GUGuGAuGuuGcucAGcucTT
2041

(115C) stab26

116
GGAGCUGAGCAACAUCACACAAG
1830
38134
rIL13: 134L21 antisense siNA
UGUGUGAuGuuGcucAGcuTT
2042

(116C) stab26

117
GAGCUGAGCAACAUCACACAAGA
1831
38135
rIL13: 135L21 antisense siNA
UUGuGuGAuGuuGcucAGcTT
2043

(117C) stab26

120
CUGAGCAACAUCACACAAGACCA
1832
38136
rIL13: 138L21 antisense siNA

GUCuuGuGuGAuGuuGcucTT
2044

(120C) stab26

121
UGAGCAACAUCACACAAGACCAG
1833
38137
rIL13: 139L21 antisense siNA

GGUcuuGuGuGAuGuuGcuTT
2045

(121C) stab26

122
GAGCAACAUCACACAAGACCAGA
1834
38138
rIL13: 140L21 antisense siNA
UGGucuuGuGuGAuGuuGcTT
2046

(122C) stab26

123
AGCAACAUCACACAAGACCAGAA
1835
38139
rIL13: 141L21 antisense siNA
CUGGucuuGuGuGAuGuuGTT
2047

(123C) stab26

124
GCAACAUCACACAAGACCAGAAG
1836
38140
rIL13: 142L21 antisense siNA
UCUGGucuuGuGuGAuGuuTT
2048

(124C) stab26

141
CAGAAGACUUCCCUGUGCAACAG
1837
38141
rIL13: 159L21 antisense siNA

GUUGcAcAGGGAAGucuucTT
2049

(141C) stab26

159
AACAGCAGCAUGGUAUGGAGCGU
1838
38142
rIL13: 177121 antisense siNA

GCUccAuAccAuGcuGcuGTT
2050

(159C) stab26

188
GACAGCUGGCGGGUUCUGUGCAG
1839
38143
rIL13: 206L21 antisense siNA

GCAcAGAAcccGccAGcuGTT
2051

(188C) stab26

217
AAUCCCUGACCAACAUCUCCAGU
1840
38144
rIL13: 235L21 antisense siNA
UGGAGAuGuuGGucAGGGATT
2052

(217C) stab26

237
AGUUGCAAUGCCAUCCACAGGAC
1841
38145
rIL13: 255L21 antisense siNA
CCUGuGGAuGGcAuuGcAATT
2053

(237C) stab26

252
CACAGGACCCAGAGGAUAUUGAA
1842
38146
rIL13: 270L21 antisense siNA
CAAuAuccucuGGGuccuGTT
2054

(252C) stab26

319
CAGAUACCAAAAUCGAAGUAGCC
1843
38147
rIL13: 337L21 antisense siNA
CUAcuucGAuuuuGGuAucTT
2055

(319C) stab26

320
AGAUACCAAAAUCGAAGUAGCCC
1844
38148
rIL13: 338L21 antisense siNA

GCUAcuucGAuuuuGGuAuTT
2056

(320C) stab26

321
GAUACCAAAAUCGAAGUAGooCA
1845
38149
rIL13: 339L21 antisense siNA

GGCuAcuucGAuuuuGGuATT
2057

(321C) stab26

322
AUACCAAAAUCGAAGUAGCCCAG
1846
38150
rIL13: 340L21 antisense siNA

GGGcuAcuucGAuuuuGGuTT
2058

(322C) stab26

323
UACCAAAAUCGAAGUAGCCCAGU
1847
38151
rIL13: 341L21 antisense siNA
UGGGcuAcuucGAuuuuGGTT
2059

(323C) stab26

360
CUCAAUUACUCCAAGCAACUUUU
1848
38152
rIL13: 378L21 antisense siNA

AAGuuGcuuGGAGuAAuuGTT
2060

(360C) stab26

361
UCAAUUACUCCAAGCAACUUUUC
1849
38153
rIL13: 379L21 antisense siNA

AAAGuuGcuuGGAGuAAuuTT
2061

(361C) stab26

362
CAAUUACUCCAAGCAACUUUUCC
1850
38154
rIL13: 380L21 antisense siNA

AAAAGuuGcuuGGAGuAAuTT
2062

(362C) stab26

375
CAACUUUUCCGCUAUGGCCACUG
1851
38155
rIL13: 393L21 antisense siNA

GUGGccAuAGcGGAAAAGuTT
2063

(375C) stab26

420
CUCAGCUGUGGACCUCAGUUGUG
1852
38156
rIL13: 438L21 antisense siNA
CAAcuGAGGuccAcAGcuGTT
2064

(420C) stab26

122
GAGCAACAUCACACAAGACCAGA
1834
39525
rIL13: 122U21 sense siNA stab00
GCAACAUCACACAAGACCATT
2065

122
GAGCAACAUCACACAAGACCAGA
1834
39526
rIL13: 140L21 antisense siNA
UGGUCUUGUGUGAUGUUGCTT
2066

(122C) stab00

120
CUGAGCAACAUCACACAAGACCA
1832
39539
rIL13: 120U21 sense siNA stab00
GAGCAACAUCACACAAGACTT
2067

321
GAUACCAAAAUCGAAGUAGCCCA
1845
39540
rIL13: 321U21 sense siNA stab00
UACCAAAAUCGAAGUAGCCTT
2068

323
UACCAAAAUCGAAGUAGCCCAGU
1847
39541
rIL13: 323U21 sense siNA stab00
CCAAAAUCGAAGUAGCCCATT
2069

120
CUGAGCAACAUCACACAAGACCA
1832
39542
rIL13: 138L21 antisense siNA
GUCUUGUGUGAUGUUGCUCTT
2070

(120C) stab00

321
GAUACCAAAAUCGAAGUAGCCCA
1845
39543
rIL13: 339L21 antisense siNA
GGCUACUUCGAUUUUGGUATT
2071

(321C) stab00

323
UACCAAAAUCGAAGUAGCCCAGU
1847
39544
rIL13: 341L21 antisense siNA
UGGGCUACUUCGAUUUUGGTT
2072

(323C) stab00

110
GCCACAGAAGUUCAGCCACCUGU
1853
38157
rIL13RA1: 110U21 sense siNA
B cAcAGAAGuucAGccAccuTT B
2073

stab07

112
CACAGAAGUUCAGCCACCUGUGA
1854
38158
rIL13RA1: 112U21 sense siNA
B cAGAAGuucAGccAccuGuTT B
2074

stab07

113
ACAGAAGUUCAGCCACCUGUGAC
1855
38159
rIL13RA1: 113U21 sense siNA
B AGAAGuucAGccAccuGuGTT B
2075

stab07

123
AGCCACCUGUGACGAAUUUGAGU
1856
38160
rIL13RA1: 123U21 sense siNA
B ccAccuGuGAcGAAuuuGATT B
2076

stab07

148
CUCUGUCGAAAAUCUCUGCACAA
1857
38161
rIL13RA1: 148U21 sense siNA
B cuGucGAAAAucucuGcAcTT B
2077

stab07

343
UGAAAGUGAGAAGCCUAGCCCUU
1858
38162
rIL13RA1: 343U21 sense siNA
B AAAGuGAGAAGccuAGcccTT B
2078

stab07

347
AGUGAGAAGCCUAGCCCUUUGGU
1859
38163
rIL13RA1: 347U21 sense siNA
B uGAGAAGccuAGcccuuuGTT B
2079

stab07

350
GAGAAGCCUAGCCCUUUGGUGAA
1860
38164
rIL13RA1: 350U21 sense siNA
B GAAGccuAGcccuuuGGuGTT B
2080

stab07

356
CCUAGCCCUUUGGUGAAAAAGUG
1861
38165
rIL13RA1: 356U21 sense siNA
B uAGcccuuuGGuGAAAAAGTT B
2081

stab07

362
CCUUUGGUGAAAAAGUGCAUCUC
1862
38166
rIL13RA1: 362U21 sense siNA
B uuuGGuGAAAAAGuGcAucTT B
2082

stab07

363
CUUUGGUGAAAAAGUGCAUCUCA
1863
38167
rIL13RA1: 363U21 sense siNA
B uuGGuGAAAAAGuGcAucuTT B
2083

stab07

365
UUGGUGAAAAAGUGCAUCUCACC
1864
38168
rIL13RA1: 365U21 sense siNA
B GGuGAAAAAGuGcAucucATT B
2084

stab07

419
GAACUGCAGUGCACUUGGCACAA
1865
38169
rIL13RA1: 419U21 sense siNA
B AcuGcAGuGcAcuuGGcAcTT B
2085

stab07

424
GCAGUGCACUUGGCACAACCUGA
1866
38170
rIL13RA1: 424U21 sense siNA
B AGuGcAcuuGGcAcAAccuTT B
2086

stab07

464
UGGCUCCCUGGAAAGAAUACAAG
1867
38171
rIL13RA1: 464U21 sense siNA
B GcucccuGGAAAGAAuAcATT B
2087

stab07

529
GGGGAAAAGUCUUCAAUGUGAAA
1868
38172
rIL13RA1: 529U21 sense siNA
B GGAAAAGucuucAAuGuGATT B
2088

stab07

585
CCUUUAAAUUGACUAAAGUGGAA
1869
38173
rIL13RA1: 585U21 sense siNA
B uuuAAAuuGAcuAAAGuGGTT B
2089

stab07

636
UAAUGGUCAAGGAUAAUGCUGGG
1870
38174
rIL13RA1: 636U21 sense siNA
B AuGGucAAGGAuAAuGcuGTT B
2090

stab07

637
AAUGGUCAAGGAUAAUGCUGGGA
1871
38175
rIL13RA1: 637U21 sense siNA
B uGGucAAGGAuAAuGcuGGTT B
2091

stab07

638
AUGGUCAAGGAUAAUGCUGGGAA
1872
38176
rIL13RA1: 638U21 sense siNA
B GGucAAGGAuAAuGcuGGGTT B
2092

stab07

640
GGUCAAGGAUAAUGCUGGGAAAA
1873
38177
rIL13RA1: 640U21 sense siNA
B ucAAGGAuAAuGcuGGGAATT B
2093

stab07

646
GGAUAAUGCUGGGAAAAUUAGGC
1874
38178
rIL13RA1: 646U21 sense siNA
B AuAAuGcuGGGAAAAuuAGTT B
2094

stab07

649
UAAUGCUGGGAAAAUUAGGCCAU
1875
38179
rIL13RA1: 649U21 sense siNA
B AuGcuGGGAAAAuuAGGccTT B
2095

stab07

650
AAUGCUGGGAAAAUUAGGCCAUC
1876
38180
rIL13RA1: 650U21 sense siNA
B uGcuGGGAAAAuuAGGccATT B
2096

stab07

654
CUGGGAAAAUUAGGCCAUCCUAC
1877
38181
rIL13RA1: 654U21 sense siNA
B GGGAAAAuuAGGccAuccuTT B
2097

stab07

733
UUUCCUCAAAAAUGGUGCCUUAU
1878
38182
rIL13RA1: 733U21 sense siNA
B uccucAAAAAuGGuGccuuTT B
2098

stab07

734
UUCCUCAAAAAUGGUGCCUUAUU
1879
38183
rIL13RA1: 734U21 sense siNA
B ccucAAAAAuGGuGccuuATT B
2099

stab07

858
AGAGGUUGAAGAGGACAAAUGCC
1880
38184
rIL13RA1: 856U21 sense siNA
B AGGuuGAAGAGGAcAAAuGTT B
2100

stab07

863
GAAGAGGACAAAUGCCAGAAUUC
1881
38185
rIL13RA1: 863U21 sense siNA
B AGAGGAcAAAuGccAGAAuTT B
2101

stab07

876
GCCAGAAUUCUGAAUUUGAUAGA
1882
38186
rIL13RA1: 876U21 sense siNA
B cAGAAuucuGAAuuuGAuATT B
2102

stab07

877
CCAGAAUUCUGAAUUUGAUAGAA
1883
38187
rIL13RA1: 877U21 sense siNA
B AGAAuucuGAAuuuGAuAGTT B
2103

stab07

890
UUUGAUAGAAACAUGGAGGGUGC
1884
38188
rIL13RA1: 890U21 sense siNA
B uGAuAGAAAcAuGGAGGGuTT B
2104

stab07

1008
UGUGGAGUAAUUGGAGCGAAGCG
1885
38189
rIL13RA1: 1008U21 sense siNA
B uGGAGuAAuuGGAGcGAAGTT B
2105

stab07

1009
GUGGAGUAAUUGGAGCGAAGCGC
1886
38190
rIL13RA1: 1009U21 sense siNA
B GGAGuAAuuGGAGcGAAGcTT B
2106

stab07

1010
UGGAGUAAUUGGAGCGAAGCGCU
1887
38191
rIL13RA1: 1010U21 sense siNA
B GAGuAAuuGGAGcGAAGcGTT B
2107

stab07

1137
GGCUUAAGAUCAUUAUAUUUCCU
1888
38192
rIL13RA1: 1137U21 sense siNA
B cuuAAGAucAuuAuAuuucTT B
2108

stab07

1153
AUUUCCUCCAAUUCCUGAUCCUG
1889
38193
rIL13RA1: 1153U21 sense siNA
B uuccuccAAuuccuGAuccTT B
2109

stab07

1161
CAAUUCCUGAUCCUGGCAAGAUU
1890
38194
rIL13RA1: 1161U21 sense siNA
B AuuccuGAuccuGGcAAGATT B
2110

stab07

1163
AUUCCUGAUCCUGGCAAGAUUUU
1891
38195
rIL13RA1: 1163U21 sense siNA
B uccuGAuccuGGcAAGAuuTT B
2111

stab07

1164
UUCCUGAUCCUGGCAAGAUUUUU
1892
38196
rIL13RA1: 1164U21 sense siNA
B ccuGAuccuGGcAAGAuuuTT B
2112

stab07

1172
CCUGGCAAGAUUUUUAAAGAAAU
1893
38197
rIL13RA1: 1172U21 sense siNA
B uGGcAAGAuuuuuAAAGAATT B
2113

stab07

1182
UUUUUAAAGAAAUGUUUGGAGAC
1894
38198
rIL13RA1: 1182U21 sense siNA
B uuuAAAGAAAuGuuuGGAGTT B
2114

stab07

1198
UGGAGACCAGAAUGAUGAUACCC
1895
38199
rIL13RA1: 1198U21 sense siNA
B GAGAccAGAAuGAuGAuAcTT B
2115

stab07

1199
GGAGACCAGAAUGAUGAUACCCU
1896
38200
rIL13RA1: 1199U21 sense siNA
B AGAccAGAAuGAuGAuAccTT B
2116

stab07

1202
GACCAGAAUGAUGAUAQCCUGCA
1897
38201
rIL13RA1: 1202U21 sense siNA
B ccAGAAuGAuGAuAcccuGTT B
2117

stab07

1203
ACCAGAAUGAUGAUACCCUGCAC
1898
38202
rIL13RA1: 1203U21 sense siNA
B cAGAAuGAuGAuAcccuGcTT B
2118

stab07

1204
CCAGAAUGAUGAUACCCUGCACU
1899
38203
rIL13RA1: 1204U21 sense siNA
B AGAAuGAuGAuAcccuGcATT B
2119

stab07

1208
AAUGAUGAUACCCUGCACUGGAA
1900
38204
rIL13RA1: 1208U21 sense siNA
B uGAuGAuAcccuGcAcuGGTT B
2120

stab07

110
GCCACAGAAGUUCAGCCACCUGU
1853
38205
rIL13RA1: 128L21 antisense siNA

AGGuGGcuGAAcuucuGuGTT
2121

(110C) stab26

112
CACAGAAGUUCAGCCACCUGUGA
1854
38206
rIL13RA1: 130L21 antisense siNA

ACAGGuGGcuGAAcuucuGTT
2122

(112C) stab26

113
ACAGAAGUUCAGCCACCUGUGAC
1855
38207
rIL13RA1: 131L21 antisense siNA
CACAGGuGGcuGAAcuucuTT
2123

(113C) stab26

123
AGCCACCUGUGACGAAUUUGAGU
1856
38208
rIL13RA1: 141L21 antisense siNA
UCAAAuucGucAcAGGuGGTT
2124

(123C) stab26

148
CUCUGUCGAAAAUCUCUGCACAA
1857
38209
rIL13RA1: 166L21 antisense siNA

GUGcAGAGAuuuucGAcAGTT
2125

(148C) stab26

343
UGAAAGUGAGAAGCCUAGCCCUU
1858
38210
rIL13RA1: 361L21 antisense siNA

GGGcuAGGcuucucAcuuuTT
2126

(343C) stab26

347
AGUGAGAAGCCUAGCCCUUUGGU
1859
38211
rIL13RA1: 366L21 antisense siNA
CAAAGGGcuAGGcuucucATT
2127

(347C) stab26

350
GAGAAGCCUAGCCCUUUGGUGAA
1860
38212
rIL13RA1: 368L21 antisense siNA
CACcAAAGGGcuAGGcuucTT
2128

(350C) stab26

356
CCUAGCCCUUUGGUGAAAAAGUG
1861
38213
rIL13RA1: 374L21 antisense siNA
CUUuuucAccAAAGGGcuATT
2129

(356C) stab26

362
CCUUUGGUGAAAAAGUGCAUCUC
1862
38214
rIL13RA1: 380L21 antisense siNA

GAUGcAcuuuuucAccAAATT
2130

(362C) stab26

363
CUUUGGUGAAAAAGUGCAUCUCA
1863
38215
rIL13RA1: 381L21 antisense siNA

AGAuGcAcuuuuucAccAATT
2131

(363C) stab26

365
UUGGUGAAAAAGUGCAUCUCACC
1864
38216
rIL13RA1: 383L21 antisense siNA
UGAGAuGcAcuuuuucAccTT
2132

(365C) stab26

419
GAACUGCAGUGCACUUGGCACAA
1865
38217
rIL13RA1: 437L21 antisense siNA

GUGccAAGuGcAcuGcAGuTT
2133

(419C) stab26

424
GCAGUGCACUUGGCACAACCUGA
1866
38218
rIL13RA1: 442L21 antisense siNA

AGGuuGuGccAAGuGcAcuTT
2134

(424C) stab26

464
UGGCUCCCUGGAAAGAAUACAAG
1867
38219
rIL13RA1: 482L21 antisense siNA
UGUAuucuuuccAGGGAGcTT
2135

(464C) stab26

529
GGGGAAAAGUCUUCAAUGUGAAA
1868
38220
rIL13RA1: 547L21 antisense siNA
UCAcAuuGAAGAcuuuuccTT
2136

(529C) stab26

585
CCUUUAAAUUGACUAAAGUGGAA
1869
38221
rIL13RA1: 603L21 antisense siNA
CCAcuuuAGucAAuuuAAATT
2137

(585C) stab26

636
UAAUGGUCAAGGAUAAUGCUGGG
1870
38222
rIL13RA1: 654L21 antisense siNA
CAGcAuuAuccuuGAccAuTT
2138

(636C) stab26

637
AAUGGUCAAGGAUAAUGCUGGGA
1871
38223
rIL13RA1: 655L21 antisense siNA
CCAGcAuuAuccuuGAccATT
2139

(637C) stab26

638
AUGGUCAAGGAUAAUGCUGGGAA
1872
38224
rIL13RA1: 656L21 antisense siNA
CCCAGcAuuAuccuuGAccTT
2140

(638C) stab26

640
GGUCAAGGAUAAUGCUGGGAAAA
1873
38225
rIL13RA1: 658L21 antisense siNA
UUCccAGcAuuAuccuuGATT
2141

(640C) stab26

646
GGAUAAUGCUGGGAAAAUUAGGC
1874
38226
rIL13RA1: 664L21 antisense siNA
CUAAuuuucccAGcAuuAuTT
2142

(646C) stab26

649
UAAUGCUGGGAAAAUUAGGCCAU
1875
38227
rIL13RA1: 667L21 antisense siNA

GGCcuAAuuuucccAGcAuTT
2143

(649C) stab26

650
AAUGCUGGGAAAAUUAGGCCAUC
1876
38228
rIL13RA1: 668L21 antisense siNA
UGGccuAAuuuucccAGcATT
2144

(650C) stab26

654
CUGGGAAAAUUAGGCCAUCCUAC
1877
38229
rIL13RA1: 672L21 antisense siNA

AGGAuGGccuAAuuuucccTT
2145

(654C) stab26

733
UUUCCUCAAAAAUGGUGCCUUAU
1878
38230
rIL13RA1: 751L21 antisense siNA

AAGGcAccAuuuuuGAGGATT
2146

(733C) stab26

734
UUCCUCAAAAAUGGUGCCUUAUU
1879
38231
rIL13RA1: 752L21 antisense siNA
UAAGGcAccAuuuuuGAGGTT
2147

(734C) stab26

856
AGAGGUUGAAGAGGACAAAUGCC
1880
38232
rIL13RA1: 874L21 antisense siNA
CAUuuGuccucuucAAccuTT
2148

(856C) stab26

863
GAAGAGGACAAAUGCCAGAAUUC
1881
38233
rIL13RA1: 881L21 antisense siNA

AUUcuGGcAuuuGuccucuTT
2149

(863C) stab26

876
GCCAGAAUUCUGAAUUUGAUAGA
1882
38234
rIL13RA1: 894L21 antisense siNA
UAUcAAAuucAGAAuucuGTT
2150

(876C) stab26

877
CCAGAAUUCUGAAUUUGAUAGAA
1883
38235
rIL13RA1: 895L21 antisense siNA
CUAucAAAuucAGAAuucuTT
2151

(877C) stab26

890
UUUGAUAGAAACAUGGAGGGUGC
1884
38236
rIL13RA1: 908L21 antisense siNA

ACCcuccAuGuuucuAucATT
2152

(890C) stab26

1008
UGUGGAGUAAUUGGAGCGAAGCG
1885
38237
rIL13RA1: 1026L21 antisense siNA
CUUcGcuccAAuuAcuccATT
2153

(1008C) stab26

1009
GUGGAGUAAUUGGAGCGAAGCGC
1886
38238
rIL13RA1: 1027L21 antisense siNA

GCUucGcuccAAuuAcuccTT
2154

(1009C) stab26

1010
UGGAGUAAUUGGAGCGAAGCGCU
1887
38239
rIL13RA1: 1028L21 antisense siNA
CGCuucGcuccAAuuAcucTT
2155

(1010C) stab26

1137
GGCUUAAGAUCAUUAUAUUUCCU
1888
38240
rIL13RA1: 1155L21 antisense siNA

GAAAuAuAAuGAucuuAAGTT
2156

(1137C) stab26

1153
AUUUCCUCCAAUUCCUGAUCCUG
1889
38241
rIL13RA1: 1171L21 antisense siNA

GGAucAGGAAuuGGAGGAATT
2157

(1153C) stab26

1161
CAAUUCCUGAUCCUGGCAAGAUU
1890
38242
rIL13RA1: 1179L21 antisense siNA
UCUuGccAGGAucAGGAAuTT
2158

(1161C) stab26

1163
AUUCCUGAUCCUGGCAAGAUUUU
1891
38243
rIL13RA1: 1181L21 antisense siNA

AAUcuuGccAGGAucAGGATT
2159

(1163C) stab26

1164
UUCCUGAUCCUGGCAAGAUUUUU
1892
38244
rIL13RA1: 1182L21 antisense siNA

AAAucuuGccAGGAucAGGTT
2160

(1164C) stab26

1172
CCUGGCAAGAUUUUUAAAGAAAU
1893
38245
rIL13RA1: 1190L21 antisense siNA
UUCuuuAAAAAucuuGccATT
2161

(1172C) stab26

1182
UUUUUAAAGAAAUGUUUGGAGAC
1894
38246
rIL13RA1: 1200L21 antisense siNA
CUCcAAAcAuuucuuuAAATT
2162

(1182C) stab26

1198
UGGAGACCAGAAUGAUGAUACCC
1895
38247
rIL13RA1: 1216L21 antisense siNA

GUAucAucAuucuGGucucTT
2163

(1198C) stab26

1199
GGAGACCAGAAUGAUGAUACCCU
1896
38248
rIL13RA1: 1217L21 antisense siNA

GGUAucAucAuucuGGucuTT
2164

(1199C) stab26

1202
GACCAGAAUGAUGAUACCCUGCA
1897
38249
rIL13RA1: 1220L21 antisense siNA
CAGGGuAucAucAuucuGGTT
2165

(1202C) stab26

1203
ACCAGAAUGAUGAUACCCUGCAC
1898
38250
rIL13RA1: 1221L21 antisense siNA

GCAGGGuAucAucAuucuGTT
2166

(1203C) stab26

1204
CCAGAAUGAUGAUACCCUGCACU
1899
38251
rIL13RA1: 1222L21 antisense siNA
UGCAGGGuAucAucAuucuTT
2167

(1204C) stab26

1208
AAUGAUGAUACCCUGCACUGGAA
1900
38252
rIL13RA1: 1226L21 antisense siNA
CCAGuGcAGGGuAucAucATT
2168

(1208C) stab26

1163
AUUCCUGAUCCUGGCAAGAUUUU
1891
39545
rIL13RA1: 1163U21 sense siNA
UCCUGAUCCUGGCAAGAUUTT
2169

stab00

1163
AUUCCUGAUCCUGGCAAGAUUUU
1891
39546
rIL13RA1: 1181L21 antisense
AAUCUUGCCAGGAUCAGGATT
2170

siNA (1163C) stab00

21
AGAGAGCUAUUGAUGGGUCUCAG
1901
37805
rIL4: 21U21 sense siNA stab07
B AGAGcuAuuGAuGGGucucTT B
2171

22
GAGAGCUAUUGAUGGGUCUCAGC
1902
37806
rIL4: 22U21 sense siNA stab07
B GAGcuAuuGAuGGGucucATT B
2172

69
UGCUUUCUCAUAUGUACCGGGAA
1903
37807
rIL4: 69U21 sense siNA stab07
B cuuucucAuAuGuAccGGGTT B
2173

75
CUCAUAUGUACCGGGAACGGUAU
1904
37808
rIL4: 75U21 sense siNA stab07
B cAuAuGuAccGGGAAcGGuTT B
2174

94
GUAUCCACGGAUGUAACGACAGC
1905
37809
rIL4: 94U21 sense siNA stab07
B AuccAcGGAuGuAAcGAcATT B
2175

103
GAUGUAACGACAGCCCUCUGAGA
1906
37810
rIL4: 103U21 sense siNA stab07
B uGuAAcGAcAGcccucuGATT B
2176

108
AACGACAGCCCUCUGAGAGAGAU
1907
37811
rIL4: 108U21 sense siNA stab07
B cGAcAGcccucuGAGAGAGTT B
2177

144
AACCAGGUCACAGAAAAAGGGAC
1908
37812
rIL4: 144U21 sense siNA stab07
B ccAGGucAcAGAAAAAGGGTT B
2178

146
CCAGGUCACAGAAAAAGGGACUC
1909
37813
rIL4: 146U21 sense siNA stab07
B AGGucAcAGAAAAAGGGAcTT B
2179

148
AGGUCACAGAAAAAGGGACUCCA
1910
37814
rIL4: 148U21 sense siNA stab07
B GucAcAGAAAAAGGGAcucTT B
2180

160
AAGGGACUCCAUGCACCGAGAUG
1911
37815
rIL4: 160U21 sense siNA stab07
B GGGAcuccAuGcAccGAGATT B
2181

175
CCGAGAUGUUUGUACCAGACGUC
1912
37816
rIL4: 175U21 sense siNA stab07
B GAGAuGuuuGuAccAGAcGTT B
2182

176
CGAGAUGUUUGUACCAGACGUCC
1913
37817
rIL4: 176U21 sense siNA stab07
B AGAuGuuuGuAccAGAcGuTT B
2183

190
CAGACGUCCUUACGGCAACAAGG
1914
37818
rIL4: 190U21 sense siNA stab07
B GAcGuccuuAcGGcAAcAATT B
2184

226
ACGAGCUCAUCUGCAGGGCUUCC
1915
37819
rIL4: 228U21 sense siNA stab07
B GAGcucAucuGcAGGGcuuTT B
2185

234
AUCUGCAGGGCUUCCAGGGUGCU
1916
37820
rIL4: 234U21 sense siNA stab07
B cuGcAGGGcuuccAGGGuGTT B
2186

259
GCAAAUUUUACUUCCCACGUGAU
1917
37821
rIL4: 259U21 sense siNA stab07
B AAAuuuuAcuucccAcGuGTT B
2187

271
UCCCACGUGAUGUACCUCCGUGC
1918
37822
rIL4: 271U21 sense siNA stab07
B ccAcGuGAuGuAccuccGuTT B
2188

272
CCCACGUGAUGUACCUCCGUGCU
1919
37823
rIL4: 272U21 sense siNA stab07
B cAcGuGAuGuAccuccGuGTT B
2189

283
UACCUCCGUGCUUGAAGAACAAG
1920
37824
rIL4: 283U21 sense siNA stab07
B ccuccGuGcuuGAAGAAcATT B
2190

379
UGAAUGAGUCCACGCUCACAACA
1921
37825
rIL4: 379U21 sense siNA stab07
B AAuGAGuccAcGcucAcAATT B
2191

398
AACACUGAAAGACUUCCUGGAAA
1922
37826
rIL4: 398U21 sense siNA stab07
B cAcuGAAAGAcuuccuGGATT B
2192

399
ACACUGAAAGACUUCCUGGAAAG
1923
37827
rIL4: 399U21 sense siNA stab07
B AcuGAAAGAcuuccuGGAATT B
2193

400
CACUGAAAGACUUCCUGGAAAGC
1924
37828
rIL4: 400U21 sense siNA stab07
B cuGAAAGAcuuccuGGAAATT B
2194

401
ACUGAAAGACUUCCUGGAAAGCC
1925
37829
rIL4: 401U21 sense siNA stab07
B uGAAAGAcuuccuGGAAAGTT B
2195

402
CUGAAAGACUUCCUGGAAAGCCU
1926
37830
rIL4: 402U21 sense siNA stab07
B GAAAGAcuuccuGGAAAGcTT B
2196

403
UGAAAGACUUCCUGGAAAGCCUA
1927
37831
rIL4: 403U21 sense siNA stab07
B AAAGAcuuccuGGAAAGccTT B
2197

404
GAAAGACUUCCUGGAAAGCCUAA
1928
37832
rIL4: 404U21 sense siNA stab07
B AAGAcuuccuGGAAAGccuTT B
2198

405
AAAGACUUCCUGGAAAGCCUAAA
1929
37833
rIL4: 405U21 sense siNA stab07
B AGAcuuccuGGAAAGccuATT B
2199

406
AAGACUUCCUGGAAAGCCUAAAA
1930
37834
rIL4: 406U21 sense siNA stab07
B GAcuuccuGGAAAGccuAATT B
2200

407
AGACUUCCUGGAAAGCCUAAAAA
1931
37835
rIL4: 407U21 sense siNA stab07
B AcuuccuGGAAAGccuAAATT B
2201

422
CCUAAAAAGCAUCCUACGAGGGA
1932
37836
rIL4: 422U21 sense siNA stab07
B uAAAAAGcAuccuAcGAGGTT B
2202

21
AGAGAGCUAUUGAUGGGUCUCAG
1901
37837
rIL4: 39L21 antisense siNA

GAGAcccAucAAuAGcucuTT
2203

(21C) stab26

22
GAGAGCUAUUGAUGGGUCUCAGC
1902
37838
rIL4: 40L21 antisense siNA
UGAGAcccAucAAuAGcucTT
2204

(22C) stab26

69
UGCUUUCUCAUAUGUACCGGGAA
1903
37839
rIL4: 87L21 antisense siNA
CCCGGuAcAuAuGAGAAAGTT
2205

(69C) stab26

75
CUCAUAUGUACCGGGAACGGUAU
1904
37840
rIL4: 93L21 antisense siNA

ACCGuucccGGuAcAuAuGTT
2206

(75C) stab26

94
GUAUCCACGGAUGUAACGACAGC
1905
37841
rIL4: 112L21 antisense siNA
UGUcGuuAcAuccGuGGAuTT
2207

(94C) stab26

103
GAUGUAACGACAGCCCUCUGAGA
1906
37842
rIL4: 121L21 antisense siNA
UCAGAGGGcuGucGuuAcATT
2208

(103C) stab26

108
AACGACAGCCCUCUGAGAGAGAU
1907
37843
rIL4: 126L21 antisense siNA
CUCucucAGAGGGcuGucGTT
2209

(108C) stab26

144
AACCAGGUCACAGAAAAAGGGAC
1908
37844
rIL4: 162L21 antisense siNA
CCCuuuuucuGuGAccuGGTT
2210

(144C) stab26

146
CCAGGUCACAGAAAAAGGGACUC
1909
37845
rIL4: 164L21 antisense siNA

GUCccuuuuucuGuGAccuTT
2211

(146C) stab26

148
AGGUCACAGAAAAAGGGACUCCA
1910
37846
rIL4: 166L21 antisense siNA

GAGucccuuuuucuGuGAcTT
2212

(148C) stab26

160
AAGGGACUCCAUGCACCGAGAUG
1911
37847
rIL4: 178L21 antisense siNA
UCUcGGuGcAuGGAGucccTT
2213

(160C) stab26

175
CCGAGAUGUUUGUACCAGACGUC
1912
37848
rIL4: 193L21 antisense siNA
CGUcuGGuAcAAAcAucucTT
2214

(175C) stab26

176
CGAGAUGUUUGUACCAGACGUCC
1913
37849
rIL4: 194L21 antisense siNA

ACGucuGGuAcAAAcAucuTT
2215

(176C) stab26

190
CAGACGUCCUUACGGCAACAAGG
1914
37850
rIL4: 208L21 antisense siNA
UUGuuGccGuAAGGAcGucTT
2216

(190C) stab26

226
ACGAGCUCAUCUGCAGGGCUUCC
1915
37851
rIL4: 244L21 antisense siNA

AAGcccuGcAGAuGAGcucTT
2217

(226C) stab26

234
AUCUGCAGGGCUUCCAGGGUGCU
1916
37852
rIL4: 252L21 antisense siNA
CACccuGGAAGcccuGcAGTT
2218

(234C) stab26

259
GCAAAUUUUACUUCCCACGUGAU
1917
37853
rIL4: 277L21 antisense siNA
CACGuGGGAAGuAAAAuuuTT
2219

(259C) stab26

271
UCCCACGUGAUGUACCUCCGUGC
1918
37854
rIL4: 289L21 antisense siNA

ACGGAGGuAcAucAcGuGGTT
2220

(271C) stab26

272
CCCACGUGAUGUACCUCCGUGCU
1919
37855
rIL4: 290L21 antisense siNA
CACGGAGGuAcAucAcGuGTT
2221

(272C) stab26

283
UACCUCCGUGCUUGAAGAACAAG
1920
37856
rIL4: 301L21 antisense siNA
UGUucuucAAGcAcGGAGGTT
2222

(283C) stab26

379
UGAAUGAGUCCACGCUCACAACA
1921
37857
rIL4: 397L21 antisense siNA
UUGuGAGcGuGGAcucAuuTT
2223

(379C) stab26

398
AACACUGAAAGACUUCCUGGAAA
1922
37858
rIL4: 416L21 antisense siNA
UCCAGGAAGucuuucAGuGTT
2224

(398C) stab26

399
ACACUGAAAGACUUCCUGGAAAG
1923
37859
rIL4: 417L21 antisense siNA
UUCcAGGAAGucuuucAGuTT
2225

(399C) stab26

400
CAGUGAAAGACUUCCUGGAAAGC
1924
37860
rIL4: 418L21 antisense siNA
UUUccAGGAAGucuuucAGTT
2226

(400C) stab26

401
ACUGAAAGACUUCCUGGAAAGCC
1925
37861
rIL4: 419L21 antisense siNA
CUUuccAGGAAGucuuucATT
2227

(401C) stab26

402
CUGAAAGACUUCCUGGAAAGCCU
1926
37862
rIL4: 420L21 antisense siNA

GCUuuccAGGAAGucuuucTT
2228

(402C) stab26

403
UGAAAGACUUCCUGGAAAGCCUA
1927
37863
rIL4: 421L21 antisense siNA

GGCuuuccAGGAAGucuuuTT
2229

(403C) stab26

404
GAAAGACUUCCUGGAAAGCCUAA
1928
37864
rIL4: 422L21 antisense siNA

AGGcuuuccAGGAAGucuuTT
2230

(404C) stab26

405
AAAGACUUCCUGGAAAGCCUAAA
1929
37865
rIL4: 423L21 antisense siNA
UAGGcuuuccAGGAAGucuTT
2231

(405C) stab26

406
AAGACUUCCUGGAAAGCCUAAAA
1930
37866
rIL4: 424L21 antisense siNA
UUAGGcuuuccAGGAAGucTT
2232

(406C) stab26

407
AGACUUCCUGGAAAGCCUAAAAA
1931
37867
rIL4: 425L21 antisense siNA
UUUAGGcuuuccAGGAAGuTT
2233

(407C) stab26

422
CCUAAAAAGCAUCCUACGAGGGA
1932
37868
rIL4: 440L21 antisense siNA
CCUcGuAGGAuGcuuuuuATT
2234

(422C) stab26

400
CACUGAAAGACUUCCUGGAAAGC
1924
39523
rIL4: 400U21 sense siNA stab00
CUGAAAGACUUCCUGGAAATT
2235

400
CACUGAAAGACUUCCUGGAAAGC
1924
39524
rIL4: 418L21 antisense siNA
UUUCCAGGAAGUCUUUCAGTT
2236

(400C) stab00

22
GAGAGCUAUUGAUGGGUCUCAGC
1902
39533
rIL4: 22U21 sense siNA stab00
GAGCUAUUGAUGGGUCUCATT
2237

404
GAAAGACUUCCUGGAAAGCCUAA
1928
39534
rIL4: 404U21 sense siNA stab00
AAGACUUCCUGGAAAGCCUTT
2238

405
AAAGACUUCCUGGAAAGCCUAAA
1929
39535
rIL4: 405U21 sense siNA stab00
AGACUUCCUGGAAAGCCUATT
2239

22
GAGAGCUAUUGAUGGGUCUCAGC
1902
39536
rIL4: 40L21 antisense siNA
UGAGACCCAUCAAUAGCUCTT
2240

(22C) stab00

404
GAAAGACUUCCUGGAAAGCCUAA
1928
39537
rIL4: 422L21 antisense siNA
AGGCUUUCCAGGAAGUCUUTT
2241

(404C) stab00

405
AAAGACUUCCUGGAAAGCCUAAA
1929
39538
rIL4: 423L21 antisense siNA
UAGGCUUUCCAGGAAGUCUTT
2242

(405C) stab00

272
ACCCCACCUGCUUCUCUGACUAC
1933
37869
rIL4R: 272U21 sense siNA stab07
B cccAccuGcuucucuGAcuTT B
2243

274
CCCACCUGCUUCUCUGACUACAU
1934
37870
rIL4R: 274U21 sense siNA stab07
B cAccuGcuucucuGAcuAcTT B
2244

277
ACCUGCUUCUCUGACUACAUCCG
1935
37871
rIL4R: 277U21 sense siNA stab07
B cuGcuucucuGAcuAcAucTT B
2245

278
CCUGCUUCUCUGACUACAUCCGC
1936
37872
rIL4R: 278U21 sense siNA stab07
B uGcuucucuGAcuAcAuccTT B
2246

279
CUGCUUCUCUGACUACAUCCGCA
1937
37873
rIL4R: 279U21 sense siNA stab07
B GcuucucuGAcuAcAuccGTT B
2247

280
UGCUUCUCUGACUACAUCCGCAC
1938
37874
rIL4R: 280U21 sense siNA stab07
B cuucucuGAcuAcAuccGcTT B
2248

281
GCUUCUCUGACUACAUCCGCACU
1939
37875
rIL4R: 281U21 sense siNA stab07
B uucucuGAcuAcAuccGcATT B
2249

383
UCUCUGAAAACCUCACAUGCACC
1940
37876
rIL4R: 383U21 sense siNA stab07
B ucuGAAAAccucAcAuGcATT B
2250

554
CUCCAGACAACCUCACACUCCAC
1941
37877
rIL4R: 554U21 sense siNA stab07
B ccAGAcAAccucAcAcuccTT B
2251

556
CCAGACAACCUCACACUCCACAC
1942
37878
rIL4R: 556U21 sense siNA stab07
B AGAcAAccucAcAcuccAcTT B
2252

557
CAGACAACCUCACACUCCACACC
1943
37879
rIL4R: 557U21 sense siNA stab07
B GAcAAccucAcAcuccAcATT B
2253

560
ACAACCUCACACUCCACACCAAU
1944
37880
rIL4R: 560U21 sense siNA stab07
B AAccucAcAcuccAcAccATT B
2254

561
CAACCUCACACUCCACACCAAUG
1945
37881
rIL4R: 561U21 sense siNA stab07
B AccucAcAcuccAcAccAATT B
2255

562
AACCUCACACUCCACACCAAUGU
1946
37882
rIL4R: 562U21 sense siNA stab07
B ccucAcAcuccAcAccAAuTT B
2256

563
ACCUCACACUCCACACCAAUGUC
1947
37883
rIL4R: 563U21 sense siNA stab07
B cucAcAcuccAcAccAAuGTT B
2257

564
CCUCACACUCCACACCAAUGUCU
1948
37884
rIL4R: 564U21 sense siNA stab07
B ucAcAcuccAcAccAAuGuTT B
2258

659
UGGUCAACAUCUCCAGAGAGGAC
1949
37885
rIL4R: 659U21 sense siNA stab07
B GucAAcAucuccAGAGAGGTT B
2259

660
GGUCAACAUCUCCAGAGAGGACA
1950
37886
rIL4R: 660U21 sense siNA stab07
B ucAAcAucuccAGAGAGGATT B
2260

663
CAACAUCUCCAGAGAGGACAACC
1951
37887
rIL4R: 663U21 sense siNA stab07
B AcAucuccAGAGAGGAcAATT B
2261

664
AACAUCUCCAGAGAGGACAACCC
1952
37888
rIL4R: 664U21 sense siNA stab07
B cAucuccAGAGAGGAcAAcTT B
2262

821
AGUGGAGUCCCAGCAUCACGUGG
1953
37889
rIL4R: 821U21 sense siNA stab07
B uGGAGucccAGcAucAcGuTT B
2263

832
AGCAUCACGUGGUACAACCCAAA
1954
37890
rIL4R: 832U21 sense siNA stab07
B cAucAcGuGGuAcAAcccATT B
2264

1033
AAGAUAUGGUGGGACCAGAUUCC
1955
37891
rIL4R: 1033U21 sense siNA
B GAuAuGGuGGGAccAGAuuTT B
2265

stab07

1304
UCCUCUGGCCAGAGAACGUUCAU
1956
37892
rIL4R: 1304U21 sense siNA
B cucuGGccAGAGAAcGuucTT B
2266

stab07

1305
CCUCUGGCCAGAGAACGUUCAUG
1957
37893
rIL4R: 1305U21 sense siNA
B ucuGGccAGAGAAcGuucATT B
2267

stab07

1363
CCAGUACAGAAUGUGGAGGAGGA
1958
37894
rIL4R: 1363U21 sense siNA
B AGuAcAGAAuGuGGAGGAGTT B
2268

stab07

1368
ACAGAAUGUGGAGGAGGAAGAGG
1959
37895
rIL4R: 1368U21 sense siNA
B AGAAuGuGGAGGAGGAAGATT B
2269

stab07

1410
CCUGAGCAUGUCACCUGAGAACA
1960
37896
rIL4R: 1410U21 sense siNA
B uGAGcAuGucAccuGAGAATT B
2270

stab07

1503
GCUGGGGGCUGAGAAUGGAGGCG
1961
37897
rIL4R: 1503U21 sense siNA
B uGGGGGcuGAGAAuGGAGGTT B
2271

stab07

1719
CAAUCCUGCCUACCGGAGUUUUA
1962
37898
rIL4R: 1719U21 sense siNA
B AuccuGccuAccGGAGuuuTT B
2272

stab07

1720
AAUCCUGCCUACCGGAGUUUUAG
1963
37899
rIL4R: 1720U21 sense siNA
B uccuGccuAccGGAGuuuuTT B
2273

stab07

1721
AUCCUGCCUACCGGAGUUUUAGU
1964
37900
rIL4R: 1721U21 sense siNA
B ccuGccuAccGGAGuuuuATT B
2274

stab07

1722
UCCUGCCUACCGGAGUUUUAGUG
1965
37901
rIL4R: 1722U21 sense siNA
B cuGccuAccGGAGuuuuAGTT B
2275

stab07

1723
CCUGCCUACCGGAGUUUUAGUGA
1966
37902
rIL4R: 1723U21 sense siNA
B uGccuAccGGAGuuuuAGuTT B
2276

stab07

1880
GGGAGCAGAUCCUUCACAUGAGU
1967
37903
rIL4R: 1880U21 sense siNA
B GAGcAGAuccuucAcAuGATT B
2277

stab07

1889
UCCUUCACAUGAGUGUCCUGCAG
1968
37904
rIL4R: 1889U21 sense siNA
B cuucAcAuGAGuGuccuGcTT B
2278

stab07

1955
AAGAGUUUGUGCAGGCAGUGAAG
1969
37905
rIL4R: 1955U21 sense siNA
B GAGuuuGuGcAGGcAGuGATT B
2279

stab07

2346
CAUUGUGUACUCGUCCCUCACCU
1970
37906
rIL4R: 2346U21 sense siNA
B uuGuGuAcucGucccucAcTT B
2280

stab07

2872
AGGGACUCAUUUUGCUUUCUCCC
1971
37907
rIL4R: 2872U21 sense siNA
B GGAcucAuuuuGcuuucucTT B
2281

stab07

2934
CUCUUGUUGCCCUACCUGCUCAG
1972
37908
rIL4R: 2934U21 sense siNA
B cuuGuuGcccuAccuGcucTT B
2282

stab07

3024
UCUCCAGCUGGAAGCUGGUCCUA
1973
37909
rIL4R: 3024U21 sense siNA
B uccAGcuGGAAGcuGGuccTT B
2283

stab07

3220
AAACUUGAUUGCCCAAAGUCACU
1974
37910
rIL4R: 3220U21 sense siNA
B AcuuGAuuGcccAAAGucATT B
2284

stab07

3221
AACUUGAUUGCCCAAAGUCACUG
1975
37911
rIL4R: 3221U21 sense siNA
B cuuGAuuGcccAAAGucAcTT B
2285

stab07

3250
ACCCACAUGUGGCCAGAAGCCAG
1976
37912
rIL4R: 3250U21 sense siNA
B ccAcAuGuGGccAGAAGccTT B
2286

stab07

3290
AGUGGGAUCCCAGUAAACAAACA
1977
37913
rIL4R: 3290U21 sense siNA
B uGGGAucccAGuAAAcAAATT B
2287

stab07

3425
GGCAGACUGCAGUCUGACUGCAU
1978
37914
rIL4R: 3425U21 sense siNA
B cAGAcuGcAGucuGAcuGcTT B
2288

stab07

3426
GCAGACUGCAGUCUGACUGCAUU
1979
37915
rIL4R: 3426U21 sense siNA
B AGAcuGcAGucuGAcuGcATT B
2289

stab07

3427
CAGACUGCAGUCUGACUGCAUUC
1980
37916
rIL4R: 3427U21 sense siNA
B GAcuGcAGucuGAcuGcAuTT B
2290

stab07

272
ACCCCACCUGCUUCUCUGACUAC
1933
37917
rIL4R: 290L21 antisense siNA

AGUcAGAGAAGcAGGuGGGTT
2291

(272C) stab26

274
CCCACCUGCUUCUCUGACUACAU
1934
37918
rIL4R: 292L21 antisense siNA

GUAGUcAGAGAAGcAGGuGTT
2292

(274C) stab26

277
ACCUGCUUCUCUGACUACAUCCG
1935
37919
rIL4R: 295L21 antisense siNA

GAUGuAGucAGAGAAGcAGTT
2293

(277C) stab26

278
CCUGCUUCUCUGACUACAUCCGC
1936
37920
rIL4R: 296L21 antisense siNA

GGAuGuAGucAGAGAAGcATT
2294

(278C) stab26

279
CUGCUUCUCUGACUACAUCCGCA
1937
37921
rIL4R: 297L21 antisense siNA
CGGAuGuAGucAGAGAAGcTT
2295

(279C) stab26

280
UGCUUCUCUGACUACAUCCGCAC
1938
37922
rIL4R: 298L21 antisense siNA

GCGGAuGuAGucAGAGAAGTT
2296

(280C) stab26

281
GCUUCUCUGACUACAUCCGCACU
1939
37923
rIL4R: 299L21 antisense siNA
UGCGGAuGuAGucAGAGAATT
2297

(281C) stab26

383
UCUCUGAAAACCUCACAUGCACC
1940
37924
rIL4R: 401L21 antisense siNA
UGCAuGuGAGGuuuucAGATT
2298

(383C) stab26

554
CUCCAGACAACCUCACACUCCAC
1941
37925
rIL4R: 572L21 antisense siNA

GGAGuGuGAGGuuGucuGGTT
2299

(554C) stab26

556
CCAGACAACCUCACACUCCACAC
1942
37926
rIL4R: 574L21 antisense siNA

GUGGAGuGuGAGGuuGucuTT
2300

(556C) stab26

557
CAGACAACCUCACACUCCACACC
1943
37927
rIL4R: 575L21 antisense siNA
UGUGGAGuGuGAGGuuGucTT
2301

(557C) stab26

560
ACAACCUCACACUCCACACCAAU
1944
37928
rIL4R: 578L21 antisense siNA
UGGuGuGGAGuGuGAGGuuTT
2302

(560C) stab26

561
CAACCUCACACUCCACACCAAUG
1945
37929
rIL4R: 579L21 antisense siNA
UUGGuGuGGAGuGuGAGGuTT
2303

(561C) stab26

562
AACCUCACACUCCACACCAAUGU
1946
37930
rIL4R: 580L21 antisense siNA

AUUGGuGuGGAGuGuGAGGTT
2304

(562C) stab26

563
ACCUCACACUCCACACCAAUGUC
1947
37931
rIL4R: 581L21 antisense siNA
CAUuGGuGuGGAGuGuGAGTT
2305

(563C) stab26

564
CCUCACACUCCACACCAAUGUCU
1948
37932
rIL4R: 582L21 antisense siNA

ACAuuGGuGuGGAGuGuGATT
2306

(564C) stab26

659
UGGUCAACAUCUCCAGAGAGGAC
1949
37933
rIL4R: 677L21 antisense siNA
CCUcucuGGAGAuGuuGAcTT
2307

(659C) stab26

660
GGUCAACAUCUCCAGAGAGGACA
1950
37934
rIL4R: 678L21 antisense siNA
UCCucucuGGAGAuGuuGATT
2308

(660C) stab26

663
CAACAUCUCCAGAGAGGACAACC
1951
37935
rIL4R: 681L21 antisense siNA
UUGuccucucuGGAGAuGuTT
2309

(663C) stab26

664
AACAUCUCCAGAGAGGACAACCC
1952
37936
rIL4R: 682L21 antisense siNA

GUUGuccucucuGGAGAuGTT
2310

(664C) stab26

821
AGUGGAGUCCCAGCAUCACGUGG
1953
37937
rIL4R: 839L21 antisense siNA

ACGuGAuGcuGGGAcuccATT
2311

(821C) stab26

832
AGCAUCACGUGGUACAACCCAAA
1954
37938
rOL4R: 850L21 antisense siNA
UGGGuuGuAccAcGuGAuGTT
2312

(832C) stab26

1033
AAGAUAUGGUGGGACCAGAUUCC
1955
37939
rIL4R: 1051L21 antisense siNA

AAUcuGGucccAccAuAucTT
2313

(1033C) stab26

1304
UCCUCUGGCCAGAGAACGUUCAU
1956
37940
rIL4R: 1322L21 antisense siNA

GAAcGuucucuGGccAGAGTT
2314

(1304C) stab26

1305
CCUCUGGCCAGAGAACGUUCAUG
1957
37941
rIL4R: 1323L21 antisense siNA
UGAAcGuucucuGGccAGATT
2315

(1305C) stab26

1363
CCAGUACAGAAUGUGGAGGAGGA
1958
37942
rIL4R: 1381L21 antisense siNA
CUCcuccAcAuucuGuAcuTT
2316

(1363C) stab26

1368
ACAGAAUGUGGAGGAGGAAGAGG
1959
37943
rIL4R: 1386L21 antisense siNA
UCUuccuccuccAcAuucuTT
2317

(1368C) stab26

1410
CCUGAGCAUGUCACCUGAGAACA
1960
37944
rIL4R: 1428L21 antisense siNA
UUCucAGGuGAcAuGcucATT
2318

(1410C) stab26

1503
GCUGGGGGCUGAGAAUGGAGGCG
1961
37945
rIL4R: 1521L21 antisense siNA
CCUccAuucucAGcccccATT
2319

(1503C) stab26

1719
CAAUCCUGCCUACCGGAGUUUUA
1962
37946
rIL4R: 1737L21 antisense siNA

AAAcuccGGuAGGcAGGAuTT
2320

(1719C) stab26

1720
AAUCCUGCCUACCGGAGUUUUAG
1963
37947
rIL4R: 1738L21 antisense siNA

AAAAcuccGGuAGGcAGGATT
2321

(1720C) stab26

1721
AUCCUGCCUACCGGAGUUUUAGU
1964
37948
rIL4R: 1739L21 antisense siNA
UAAAAcuccGGuAGGcAGGTT
2322

(1721C) stab26

1722
UCCUGCCUACCGGAGUUUUAGUG
1965
37949
rIL4R: 1740L21 antisense siNA
CUAAAAcuccGGuAGGcAGTT
2323

(1722C) stab26

1723
CCUGCCUACCGGAGUUUUAGUGA
1966
37950
rIL4R: 1741L21 antisense siNA

ACUAAAAcuccGGuAGGcATT
2324

(1723C) stab26

1880
GGGAGCAGAUCCUUCACAUGAGU
1967
37951
rIL4R: 1898L21 antisense siNA
UCAuGuGAAGGAucuGcucTT
2325

(1880C) stab26

1889
UCCUUCACAUGAGUGUCCUGGAG
1968
37952
rIL4R: 1907L21 antisense siNA

GCAGGAcAcucAuGuGAAGTT
2326

(1889C) stab26

1955
AAGAGUUUGUGCAGGCAGUGAAG
1969
37953
rIL4R: 1973L21 antisense siNA
UCAcuGccuGcAcAAAcucTT
2327

(1955C) stab26

2346
CAUUGUGUACUCGUCCCUCACCU
1970
37954
rIL4R: 2364L21 antisense siNA

GUGAGGGAcGAGuAcAcAATT
2328

(2346C) stab26

2872
AGGGACUCAUUUUGCUUUCUCCC
1971
37955
rIL4R: 2890L21 antisense siNA

GAGAAAGcAAAAuGAGuccTT
2329

(2872C) stab26

2934
CUCUUGUUGCCCUACCUGCUCAG
1972
37956
rIL4R: 2952L21 antisense siNA

GAGcAGGuAGGGcAAcAAGTT
2330

(2934C) stab26

3024
UCUCCAGCUGGAAGCUGGUCCUA
1973
37957
rIL4R: 3042L21 antisense siNA

GGAccAGcuuccAGcuGGATT
2331

(3024C) stab26

3220
AAACUUGAUUGCCCAAAGUCACU
1974
37958
rIL4R: 3238L21 antisense siNA
UGAcuuuGGGcAAucAAGuTT
2332

(3220C) stab26

3221
AACUUGAUUGCCCAAAGUCACUG
1975
37959
rIL4R: 3239L21 antisense siNA

GUGAcuuuGGGcAAucAAGTT
2333

(3221C) stab26

3250
ACCCACAUGUGGCCAGAAGCCAG
1976
37960
rIL4R: 3268L21 antisense siNA

GGCuucuGGccAcAuGuGGTT
2334

(3250C) stab26

3290
AGUGGGAUCCCAGUAAACAAACA
1977
37961
rIL4R: 3308L21 antisense siNA
UUUGuuuAcuGGGAucccATT
2335

(3290C) stab26

3425
GGCAGACUGCAGUCUGACUGCAU
1978
37962
rIL4R: 3443L21 antisense siNA

GCAGucAGAcuGcAGucuGTT
2336

(3425C) stab26

3426
GCAGACUGCAGUCUGACUGCAUU
1979
37963
rIL4R: 3444L21 antisense siNA
UGCAGucAGAcuGcAGucuTT
2337

(3426C) stab26

3427
CAGACUGCAGUCUGACUGCAUUC
1980
37964
rIL4R: 3445L21 antisense siNA

AUGcAGucAGAcuGcAGucTT
2338

(3427C) stab26

3220
AAACUUGAUUGCCCAAAGUCACU
1974
39527
rIL4R: 3220U21 sensesiNA
ACUUGAUUGCCCAAAGUCATT
2339

stab00

3220
AAACUUGAUUGCCCAAAGUCACU
1974
39528
rIL4R: 3238L21 antisense siNA
UGACUUUGGGCAAUCAAGUTT
2340

(3220C) stab00

Uppercase = ribonucleotide

u, c = 2′-deoxy2′-fluoro U, C

T = thymidine

B = inverted deoxy abasic

s = phosphorothloate linkage

A = deoxy Adenosine

G = deoxy Guanosine

G = 2′-O-methyl Guanosine

A = 2′-O-methyl Adenosine

h = human

r = rat

m = mouse

TABLE IV

Non-limiting examples of Stabilization Chemistries for

chemically modified siNA constructs

Chemistry
pyrimidine
Purine
cap
p = S
Strand

“Stab 00”
Ribo
Ribo
TT at 3′-ends

S/AS

“Stab 1”
Ribo
Ribo
—
5 at 5′-end
S/AS

1 at 3′-end

“Stab 2”
Ribo
Ribo
—
All linkages
Usually AS

“Stab 3”
2′-fluoro
Ribo
—
4 at 5′-end
Usually S

4 at 3′-end

“Stab 4”
2′-fluoro
Ribo
5′ and 3′-ends
—
Usually S

“Stab 5”
2′-fluoro
Ribo
—
1 at 3′-end
Usually AS

“Stab 6”
2′-O-Methyl
Ribo
5′ and 3′-
—
Usually S

ends

“Stab 7”
2′-fluoro
2′-deoxy
5′ and 3′-
—
Usually S

ends

“Stab 8”
2′-fluoro
2′-O-Methyl
—
1 at 3′-end
S/AS

“Stab 9”
Ribo
Ribo
5′ and 3′-
—
Usually S

ends

“Stab 10”
Ribo
Ribo
—
1 at 3′-end
Usually AS

“Stab 11”
2′-fluoro
2′-deoxy
—
1 at 3′-end
Usually AS

“Stab 12”
2′-fluoro
LNA
5′ and 3′-

Usually S

ends

“Stab 13”
2′-fluoro
LNA

1 at 3′-end
Usually AS

“Stab 14”
2′-fluoro
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 15”
2′-deoxy
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 16”
Ribo
2′-O-
5′ and 3′-

Usually S

Methyl
ends

“Stab 17”
2′-O-Methyl
2′-O-
5′ and 3′-

Usually S

Methyl
ends

“Stab 18”
2′-fluoro
2′-O-
5′ and 3′-

Usually S

Methyl
ends

“Stab 19”
2′-fluoro
2′-O-
3′-end

S/AS

Methyl

“Stab 20”
2′-fluoro
2′-deoxy
3′-end

Usually AS

“Stab 21”
2′-fluoro
Ribo
3′-end

Usually AS

“Stab 22”
Ribo
Ribo
3′-end

Usually AS

“Stab 23”
2′-fluoro*
2′-deoxy*
5′ and 3′-

Usually S

ends

“Stab 24”
2′-fluoro*
2′-O-
—
1 at 3′-end
S/AS

Methyl*

“Stab 25”
2′-fluoro*
2′-O-
—
1 at 3′-end
S/AS

Methyl*

“Stab 26”
2′-fluoro*
2′-O-
—

S/AS

Methyl*

“Stab 27”
2′-fluoro*
2′-O-
3′-end

S/AS

Methyl*

“Stab 28”
2′-fluoro*
2′-O-
3′-end

S/AS

Methyl*

“Stab 29”
2′-fluoro*
2′-O-

1 at 3′-end
S/AS

Methyl*

“Stab 30”
2′-fluoro*
2′-O-

S/AS

Methyl*

“Stab 31”
2′-fluoro*
2′-O-
3′-end

S/AS

Methyl*

“Stab 32”
2′-fluoro
2′-O-

S/AS

Methyl

“Stab 33”
2′-fluoro
2′-deoxy*
5′ and 3′-
—
Usually S

ends

“Stab 34”
2′-fluoro
2′-O-
5′ and 3′-

Usually S

Methyl*
ends

“Stab 3F”
2′-OCF3
Ribo
—
4 at 5′-end
Usually S

4 at 3′-end

“Stab 4F”
2′-OCF3
Ribo
5′ and 3′-
—
Usually S

ends

“Stab 5F”
2′-OCF3
Ribo
—
1 at 3′-end
Usually AS

“Stab 7F”
2′-OCF3
2′-deoxy
5′ and 3′-
—
Usually S

ends

“Stab 8F”
2′-OCF3
2′-O-
—
1 at 3′-end
S/AS

Methyl

“Stab 11F”
2′-OCF3
2′-deoxy
—
1 at 3′-end
Usually AS

“Stab 12F”
2′-OCF3
LNA
5′ and 3′-

Usually S

ends

“Stab 13F”
2′-OCF3
LNA

1 at 3′-end
Usually AS

“Stab 14F”
2′-OCF3
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 15F”
2′-OCF3
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 18F”
2′-OCF3
2′-O-
5′ and 3′-

Usually S

Methyl
ends

“Stab 19F”
2′-OCF3
2′-O-
3′-end

S/AS

Methyl

“Stab 20F”
2′-OCF3
2′-deoxy
3′-end

Usually AS

“Stab 21F”
2′-OCF3
Ribo
3′-end

Usually AS

“Stab 23F”
2′-OCF3*
2′-deoxy*
5′ and 3′-

Usually S

ends

“Stab 24F”
2′-OCF3*
2′-O-
—
1 at 3′-end
S/AS

Methyl*

“Stab 25F”
2′-OCF3*
2′-O-
—
1 at 3′-end
S/AS

Methyl*

“Stab 26F”
2′-OCF3*
2′-O-
—

S/AS

Methyl*

“Stab 27F”
2′-OCF3*
2′-O-
3′-end

S/AS

Methyl*

“Stab 28F”
2′-OCF3*
2′-O-
3′-end

S/AS

Methyl*

“Stab 29F”
2′-OCF3*
2′-O-

1 at 3′-end
S/AS

Methyl*

“Stab 30F”
2′-OCF3*
2′-O-

S/AS

Methyl*

“Stab 31F”
2′-OCF3*
2′-O-
3′-end

S/AS

Methyl*

“Stab 32F”
2′-OCF3
2′-O-

S/AS

Methyl

“Stab 33F”
2′-OCF3
2′-deoxy*
5′ and 3′-
—
Usually S

ends

“Stab 34F”
2′-OCF3
2′-O-
5′ and 3′-

Usually S

Methyl*
ends

CAP = any terminal cap, see for example FIG. 10.

All Stab 00-34 chemistries can comprise 3′-terminal thymidine (TT) residues

All Stab 00-34 chemistries typically comprise about 21 nucleotides, but can vary as described herein.

S = sense strand

AS = antisense strand

*Stab 23 has a single ribonucleotide adjacent to 3′-CAP

*Stab 24 and Stab 28 have a single ribonucleotide at 5′-terminus

*Stab 25, Stab 26, and Stab 27 have three ribonucleotides at 5′-terminus

*Stab 29, Stab 30, Stab 31, Stab 33, and Stab 34 any purine at first three nucleotide positions from 5′-terminus are ribonucleotides

p = phosphorothioate linkage

TABLE V

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

A. 2.5 μmol Synthesis Cycle ABI 394 Instrument

Phosphoramidites
6.5
163 μL
45 sec
2.5
min
7.5
min

S-Ethyl Tetrazole
23.8
238 μL
45 sec
2.5
min
7.5
min

Acetic Anhydride
100
233 μL
5 sec
5
sec
5
sec

N-Methyl Imidazole
186
233 μL
5 sec
5
sec
5
sec

TCA
176
2.3 mL
21 sec
21
sec
21
sec

Iodine
11.2
1.7 mL
45 sec
45
sec
45
sec

Beaucage
12.9
645 μL
100 sec
300
sec
300
sec

Acetonitrile
NA
6.67 mL
NA
NA
NA

B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

Phosphoramidites
15
31 μL
45 sec
233
sec
465
sec

S-Ethyl Tetrazole
38.7
31 μL
45 sec
233
min
465
sec

Acetic Anhydride
655
124 μL
5 sec
5
sec
5
sec

N-Methyl Imidazole
1245
124 μL
5 sec
5
sec
5
sec

TCA
700
732 μL
10 sec
10
sec
10
sec

Iodine
20.6
244 μL
15 sec
15
sec
15
sec

Beaucage
7.7
232 μL
100 sec
300
sec
300
sec

Acetonitrile
NA
2.64 mL
NA
NA
NA

C. 0.2 μmol Synthesis Cycle 96 well Instrument

Equivalents: DNA/
Amount: DNA/2′-O-

Wait Time* 2′-O-

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

Phosphoramidites
22/33/66
40/60/120
μL
60 sec
180
sec
360 sec

S-Ethyl Tetrazole
70/105/210
40/60/120
μL
60 sec
180
min
360 sec

Acetic Anhydride
265/265/265
50/50/50
μL
10 sec
10
sec
10 sec

N-Methyl
502/502/502
50/50/50
μL
10 sec
10
sec
10 sec

Imidazole

TCA
238/475/475
250/500/500
μL
15 sec
15
sec
15 sec

Iodine
6.8/6.8/6.8
80/80/80
μL
30 sec
30
sec
30 sec

Beaucage
34/51/51
80/120/120

100 sec
200
sec
200 sec

Acetonitrile
NA
1150/1150/1150
μL
NA
NA
NA

Wait time does not include contact time during delivery.

Tandem synthesis utilizes double coupling of linker molecule

Number	Date	Country
60358580	Feb 2002	US
60358580	Feb 2002	US
60363124	Mar 2002	US
60363124	Mar 2002	US
60386782	Jun 2002	US
60386782	Jun 2002	US
60406784	Aug 2002	US
60406784	Aug 2002	US
60408378	Sep 2002	US
60408378	Sep 2002	US
60409293	Sep 2002	US
60409293	Sep 2002	US
60440129	Jan 2003	US
60440129	Jan 2003	US
60292217	May 2001	US
60362016	Mar 2002	US
60306883	Jul 2001	US
60311865	Aug 2001	US

	Number	Date	Country
Parent	11001347	Dec 2004	US
Child	11756240		US
Parent	10922675	Aug 2004	US
Child	11001347		US
Parent	10863973	Jun 2004	US
Child	10922675		US
Parent	PCT/US03/04566	Feb 2003	US
Child	10863973		US
Parent	PCT/US04/16390	May 2004	US
Child	PCT/US03/04566		US
Parent	10826966	Apr 2004	US
Child	PCT/US04/16390		US
Parent	10757803	Jan 2004	US
Child	10826966		US
Parent	10720448	Nov 2003	US
Child	10757803		US
Parent	10693059	Oct 2003	US
Child	10720448		US
Parent	10444853	May 2003	US
Child	10693059		US
Parent	PCT/US03/05346	Feb 2003	US
Child	10444853		US
Parent	PCT/US03/05028	Feb 2003	US
Child	PCT/US03/05346		US
Parent	PCT/US04/13456	Apr 2004	US
Child	11001347		US
Parent	10780447	Feb 2004	US
Child	PCT/US04/13456		US
Parent	10427160	Apr 2003	US
Child	10780447		US
Parent	PCT/US02/15876	May 2002	US
Child	10427160		US
Parent	10727780	Dec 2003	US
Child	PCT/US02/15876		US

RNA Interference Mediated Inhibition Of Interleukin and Interleukin Gene Expression Using Short Interfering Nucleic Acid (siNA)

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Parent Case Info

Provisional Applications (18)

Continuation in Parts (17)