Multiplex Dicer Substrate Rna Interference Molecules Having Joining Sequences

Abstract

The present invention is based, at least in part, upon the insight that compound DsiRNA agents can be generated using site-specific RNase H-cleavable double stranded nucleic acid double stranded nucleic acid regions to attach, e.g., one DsiRNA moiety to another DsiRNA moiety and/or one DsiRNA moiety to a functional group and/or payload. Because such double stranded nucleic acid joining sequences are site-specifically RNase H-cleavable, the bifunctional molecule is cleaved into DsiRNAs which bear terminal ends that orient dicer cleavage. The detrimental impact of administering a single double stranded nucleic acid RNAi agent of longer than 30-35 nucleotides in length (e.g., provocation of interferon response) can be minimized, as once administered or delivered to a subject or RNase H-containing cell, RNase H cleavage produces a shortened, active DsiRNA agent(s). The invention also provides bifunctional DsiRNA agents that are joined by double stranded DNA extension joining sequences--such bifunctional DsiRNA agents joined by dsDNA sequences do not provoke RNase H cleavage.

Description

RELATED APPLICATIONS

[0001] This present invention is a U.S. Utility patent application which claims the benefit of U.S. provisional patent application 61/151,841, filed on Feb. 11, 2009, the entirety of which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] It has recently been discovered that dsRNA agents possessing strand lengths longer than 21-23 nucleotide siRNAs--specifically dsRNA agents wherein each strand is of 25 to 30, or even 35 nucleotides in length--are surprisingly effective at reducing target gene expression in mammalian cells (Rossi et al., U.S. Patent Application Nos. 2005/0244858 and US 2005/0277610). Such Dicer substrate siRNA ("DsiRNA") agents have been shown to possess enhanced potency as compared to 21-23 nucleotide siRNAs directed at the same target, e.g., DsiRNAs have been shown to be active at concentrations less than 1 nM. Certain preferred structures for DsiRNA agents have recently been described (Rossi et al., U.S. Patent Application No. 2007/0265220).

[0003] While the synthesis and use of combination RNAi therapies has been previously described (see, e.g., WO 2005/076999), such combination RNAi therapies have largely described co-delivered, unlinked RNAi agents. Indeed, in view of the immune responses associated with administration of longer dsRNAs (Stark et al., 1998 Annu Rev Biochem 67:227-264), the skilled artisan would generally not view a combination RNAi therapy composition comprising multiple RNAi agents that are joined by double-stranded nucleotides as an attractive therapeutic agent.

[0004] A tandem siRNA agent (within which each siRNA moiety is of 19 nucleotides in length) featuring an RNA:DNA linker sequence was recently disclosed (Aygun and Feinstein, U.S. Patent Application No. 2008/0293655). However, while inhibitory activity was reported for such an agent, the inhibitory efficacy observed for such a tandem siRNA agent against its best-inhibited target RNA was less than 60% at a concentration of 10 nanomolar and less than 80% at a concentration of 20 nanomolar, respectively. Such efficacy was lower than what is commonly observed for unlinked siRNA agents.

[0005] At least in view of the above, a need exists for combination RNAi therapy agents that possess enhanced efficacy and duration of effect relative to previously described tandem siRNA agents.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention is based, at least in part, upon the insight that compound DsiRNA agents can be generated using RNase H-cleavable double stranded nucleic acid regions to attach, e.g., one DsiRNA moiety to another DsiRNA moiety and/or one DsiRNA moiety to a functional group and/or payload. Because such double stranded nucleic acid joining sequences are RNase H-cleavable, the detrimental impact of administering a single double stranded nucleic acid RNAi agent of longer than 30-35 nucleotides in length (e.g., provocation of interferon response) can be minimized, as once administered or delivered to a subject or RNase H-containing cell, RNase H cleavage produces a shortened, active DsiRNA agent(s).

[0007] The instant invention is also based, at least in part, upon the insight that effective compound DsiRNA agents can be generated using double stranded DNA:DNA extension regions, or even a single stranded DNA region, to attach one DsiRNA moiety to another DsiRNA moiety.

[0008] In one aspect, the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 35 ribonucleotides in length; a second region possessing a first oligonucleotide strand and a second oligonucleotide strand having a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of the second region in an RNase H cleavage assay, where the second region is covalently attached to the first region by an attachment that is either a covalent bond connecting the most 5' nucleotide of the first strand of the second region to the most 3' nucleotide of the first strand of the first region or a covalent bond connecting the most 3' nucleotide of the second oligonucleotide strand of the second region to the most 5' nucleotide of the second oligonucleotide strand of the first region, or both; and a third region possessing a first oligonucleotide strand possessing ribonucleotides and having a 3' terminus and a second oligonucleotide strand possessing ribonucleotides and having a 5' terminus, where the third region is covalently attached to the second region by an attachment that is either a covalent bond connecting the most 5' nucleotide of the first strand of the third region to the most 3' nucleotide of the first strand of the second region or a covalent bond connecting the most 3' nucleotide of the second oligonucleotide strand of the third region to the most 5' nucleotide of the second oligonucleotide strand of the second region, or both, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 35 ribonucleotides in length.

[0009] In another aspect, the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 35 ribonucleotides in length; a second region possessing a first oligonucleotide strand and a second oligonucleotide strand having a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of the second region in an RNase H cleavage assay, where the first oligonucleotide strand of the second region has deoxyribonucleotides and a 3' terminus and the most 5' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the first region and the most 3' nucleotide of the second oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the second oligonucleotide strand of the first region; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a 3' terminus, where the 5' terminus of the first oligonucleotide strand of the third region is located immediately adjacent to the 3' terminus of the first oligonucleotide strand of the second region, and a second oligonucleotide strand having ribonucleotides and having a 5' terminus, where the most 3' nucleotide of the second oligonucleotide strand of the third region is covalently attached to the most 5' residue of the second oligonucleotide strand of the second region, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 35 ribonucleotides in length.

[0010] In an additional aspect, the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 35 ribonucleotides in length; a second region possessing a first oligonucleotide strand and a second oligonucleotide strand having a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of the second region in an RNase H cleavage assay, where the most 5' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the first region and the most 3' nucleotide of the second oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the second oligonucleotide strand of the first region and where the second oligonucleotide strand of the second region has deoxyribonucleotides and a 5' terminus; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 3' terminus, where the most 5' nucleotide of the first oligonucleotide strand of the third region is covalently attached to the most 3' residue of the first oligonucleotide strand of the second region, and a second oligonucleotide strand having ribonucleotides and having a 5' terminus and a 3' terminus, where the 3' terminus of the second oligonucleotide strand of the third region is located immediately adjacent to the 5' terminus of the second oligonucleotide strand of the second region, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 35 ribonucleotides in length.

[0011] In another aspect, the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a 3' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; a second region possessing a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of the second region in an RNase H cleavage assay, where the RNA:DNA duplex has a first oligonucleotide strand having deoxyribonucleotides and a 5' terminus, where the 5' terminus of the first oligonucleotide strand of the second region is located immediately adjacent to the 3' terminus of the first oligonucleotide strand of the first region, and a second oligonucleotide strand, where the most 3' nucleotide of the second oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the second oligonucleotide strand of the first region; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 3' terminus, where the most 5' nucleotide of the first oligonucleotide strand of the third region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the second region, and a second oligonucleotide strand having ribonucleotides and having a 5' terminus, where the most 3' nucleotide of the second oligonucleotide strand of the third region is covalently attached to the most 5' residue of the second oligonucleotide strand of the second region, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 30 ribonucleotides in length.

[0012] In a further aspect, the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 5' terminus and a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; a second region possessing a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of the second region in an RNase H cleavage assay, where the RNA:DNA duplex has a first oligonucleotide strand, where the most 5' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the first region, and a second oligonucleotide strand having deoxyribonucleotides and a 3' terminus, where the 3' terminus of the second oligonucleotide strand of the second region is located immediately adjacent to but is not covalently attached to the 5' terminus of the second oligonucleotide strand of the first region; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 3' terminus, where the most 3' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the first oligonucleotide strand of the third region, and a second oligonucleotide strand having ribonucleotides and having a 5' terminus, where the most 3' nucleotide of the second oligonucleotide strand of the third region is covalently attached to the most 5' residue of the second oligonucleotide strand of the second region, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 30 ribonucleotides in length.

[0013] In an additional aspect, the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 25 and 35 nucleotides in length; a second region possessing a first oligonucleotide strand and a second oligonucleotide strand having a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of the second region in an RNase H cleavage assay, where the second region is covalently attached to the first region by an attachment that is either a covalent bond connecting the most 5' nucleotide of the first strand of the second region to the most 3' nucleotide of the first strand of the first region or a covalent bond connecting the most 3' nucleotide of the second oligonucleotide strand of the second region to the most 5' nucleotide of the second oligonucleotide strand of the first region, or both; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 3' terminus and a second oligonucleotide strand having ribonucleotides and having a 5' terminus, where the third region is covalently attached to the second region by an attachment that is either a covalent bond connecting the most 5' nucleotide of the first strand of the third region to the most 3' nucleotide of the first strand of the second region or a covalent bond connecting the most 3' nucleotide of the second oligonucleotide strand of the third region to the most 5' nucleotide of the second oligonucleotide strand of the second region, or both, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 35 ribonucleotides in length.

[0014] In one embodiment, the RNase H cleavage assay is an in vitro RNase H cleavage assay. In another embodiment, the RNase H cleavage assay is a mammalian cell RNase H cleavage assay.

[0015] In an additional embodiment, the first oligonucleotide strand of the second region has between four and twenty deoxyribonucleotides that base pair with ribonucleotides of the second oligonucleotide strand of the second region to form an RNA:DNA duplex.

[0016] Another aspect of the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 35 nucleotides in length; a second region possessing a RNA:DNA duplex, where the RNA:DNA duplex has a first oligonucleotide strand having a 3' terminus, where the most 5' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the first region, and a second oligonucleotide strand, where the most 3' nucleotide of the second oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the second oligonucleotide strand of the first region, where the first oligonucleotide strand of the second region has between four and forty deoxyribonucleotides that form a RNA:DNA duplex with ribonucleotides of the second oligonucleotide strand of the second region; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a 3' terminus, where the 5' terminus of the first oligonucleotide strand of the third region is located immediately adjacent to the 3' terminus of the first oligonucleotide strand of the second region, and a second oligonucleotide strand having ribonucleotides and having a 5' terminus, where the most 3' nucleotide of the second oligonucleotide strand of the third region is covalently attached to the most 5' residue of the second oligonucleotide strand of the second region, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 35 ribonucleotides in length.

[0017] An additional aspect of the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 35 nucleotides in length; a second region possessing a RNA:DNA duplex, where the RNA:DNA duplex has a first oligonucleotide strand, where the most 5' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the first region, and a second oligonucleotide strand having a 5' terminus, where the most 3' nucleotide of the second oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the second oligonucleotide strand of the first region, where the second oligonucleotide strand of the second region has between four and twenty deoxyribonucleotides that form a RNA:DNA duplex with ribonucleotides of the first oligonucleotide strand of the second region; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 3' terminus, where the most 5' nucleotide of the first oligonucleotide strand of the third region is covalently attached to the most 3' residue of the first oligonucleotide strand of the second region, and a second oligonucleotide strand having ribonucleotides and having a 5' terminus and a 3' terminus, where the 3' terminus of the second oligonucleotide strand of the third region is located immediately adjacent to the 5' terminus of the second oligonucleotide strand of the second region, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 35 ribonucleotides in length.

[0018] A further aspect of the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a 3' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 35 nucleotides in length; a second region possessing a RNA:DNA duplex, where the RNA:DNA duplex has a first oligonucleotide strand having a 5' terminus, where the 5' terminus of the first oligonucleotide strand of the second region is located immediately adjacent to the 3' terminus of the first oligonucleotide strand of the first region, and a second oligonucleotide strand, where the most 3' nucleotide of the second oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the second oligonucleotide strand of the first region, where the first oligonucleotide strand of the second region has between four and twenty deoxyribonucleotides that base pair with ribonucleotides of the second oligonucleotide strand of the second region to form an RNA:DNA duplex; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 3' terminus, where the most 5' nucleotide of the first oligonucleotide strand of the third region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the second region, and a second oligonucleotide strand having ribonucleotides and having a 5' terminus, where the most 3' nucleotide of the second oligonucleotide strand of the third region is covalently attached to the most 5' residue of the second oligonucleotide strand of the second region, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 35 ribonucleotides in length.

[0019] Another aspect of the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 5' terminus and a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 35 nucleotides in length; a second region possessing a RNA:DNA duplex, where the RNA:DNA duplex has a first oligonucleotide strand, where the most 5' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the first region, and a second oligonucleotide strand having a 3' terminus, where the 3' terminus of the second oligonucleotide strand of the second region is located immediately adjacent to but is not covalently attached to the 5' terminus of the second oligonucleotide strand of the first region, where the second oligonucleotide strand of the second region has between four and twenty deoxyribonucleotides that form a RNA:DNA duplex with ribonucleotides of the first oligonucleotide strand of the second region; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 3' terminus, where the most 3' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the first oligonucleotide strand of the third region, and a second oligonucleotide strand having ribonucleotides and having a 5' terminus, where the most 3' nucleotide of the second oligonucleotide strand of the third region is covalently attached to the most 5' residue of the second oligonucleotide strand of the second region, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 35 ribonucleotides in length.

[0020] In one embodiment, the first region possesses a duplex of at least 25 nucleotides in length. In another embodiment, the third region possesses a duplex of at least 25 nucleotides in length. In an additional embodiment, the second oligonucleotide strand of the first region is sufficiently complementary to a first target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target gene expression when the nucleic acid duplex is introduced into a mammalian cell. In another embodiment, the first or second strand of the third region is sufficiently complementary to a second target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target gene expression when the nucleic acid duplex is introduced into a mammalian cell.

[0021] In one embodiment, the first target RNA or the second target RNA is K-RAS, HPRT1, VEGF, VEGFR, EGF, EGFR or an HCV target RNA sequence. Optionally, both the first and second target RNAs are K-RAS, HPRT1, VEGF, VEGFR, EGF, EGFR or an HCV target RNA sequence. In a related embodiment, paired target RNAs of the first and second regions are HPRT1 and K-RAS; VEGF and VEGFR; or EGF and EGFR.

[0022] In another embodiment, the nucleic acid duplex reduces target gene expression (of either the first target RNA, the second target RNA, or both) in a mammalian cell in vitro by an amount (expressed by %) of at least 10%, at least 50% or at least 80-90%.

[0023] In one embodiment, the second oligonucleotide strand of the first region possesses a 3' overhang of 1-4 nucleotides in length. In another embodiment, the first oligonucleotide strand of the third region possesses a 3' overhang of 1-4 nucleotides in length. In an additional embodiment the 3' overhang is 1-3 nucleotides in length, or, optionally, 1-2 nucleotides in length. In a related embodiment, the nucleotides of the 3' overhang comprise a modified nucleotide. Optionally, the modified nucleotide residue is 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino or 2'-O-(N-methlycarbamate). In another embodiment, all nucleotides of the 3' overhang are modified nucleotides. In an additional embodiment, the 3' overhang is two nucleotides in length and the modified nucleotide is a 2'-O-methyl modified ribonucleotide.

[0024] In one embodiment, the second oligonucleotide strand of the first region, starting from the nucleotide residue of the second oligonucleotide strand of the first region that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand of the first region, has unmodified nucleotide residues at all positions from position 20 to the most 5' residue of the second oligonucleotide strand of the first region. In another embodiment, the first oligonucleotide strand of the third region, starting from the nucleotide residue of the first oligonucleotide strand of the third region that is complementary to the 5' terminal nucleotide residue of the second oligonucleotide strand of the third region, has unmodified nucleotide residues at all positions from position 20 to the most 5' residue of the first oligonucleotide strand of the third region. In an additional embodiment, the second oligonucleotide strand of the first region has modified nucleotides at positions 1, 2, and 3 from the 3' terminus of the second oligonucleotide strand of the first region. In another embodiment, the first oligonucleotide strand of the third region has modified nucleotides at positions 1, 2, and 3 from the 3' terminus of the first oligonucleotide strand of the third region.

[0025] In an additional embodiment, at least the two most 3' nucleotide residues of the first oligonucleotide strand of the first region are deoxyribonucleotides that base pair with two deoxyribonucleotides of the second oligonucleotide strand of the first region. In a further embodiment, at least the two most 5' nucleotide residues of the first oligonucleotide strand of the third region are deoxyribonucleotides that base pair with two deoxyribonucleotides of the second oligonucleotide strand of the third region. In another embodiment, the two most 3' nucleotide residues of the second oligonucleotide strand of the second region are modified ribonucleotides.

[0026] In one embodiment, the two most 5' nucleotide residues of the second oligonucleotide strand of the second region are modified ribonucleotides. In an additional embodiment, the second oligonucleotide strand of the third region, starting from the most 3' nucleotide residue of the second oligonucleotide strand of the third region, has alternating modified and unmodified nucleotide residues. In another embodiment, the two most 5' nucleotide residues of the first oligonucleotide strand of the second region are modified ribonucleotides. In an additional embodiment, the two most 3' nucleotide residues of the first oligonucleotide strand of the second region are modified ribonucleotides. In another embodiment, the first oligonucleotide strand of the third region, starting from the 3' terminus of the first oligonucleotide strand of the third region, has alternating modified and unmodified nucleotide residues.

[0027] In one embodiment, the 3' terminus of the first oligonucleotide strand of the third region and the 5' terminus of the second oligonucleotide strand of the third region form a blunt end. In an additional embodiment, at least one of positions 1, 2 or 3 from the 3' terminus of the 3' terminus of the first oligonucleotide strand of the third region is a deoxyribonucleotide. In another embodiment, the deoxynucleotide residues of the second region that comprise the RNA:DNA duplex are unmodified deoxyribonucleotides.

[0028] In a further embodiment, at least 50% of all deoxyribonucleotides of the nucleic acid duplex are unmodified deoxyribonucleotides. In one embodiment, the first oligonucleotide strand of the third region is attached to the second oligonucleotide strand of the third region by a nucleotide sequence, where the nucleotide sequence attaches the most 3' nucleotide of the first oligonucleotide strand of the third region that base pairs with a nucleotide of the second oligonucleotide strand of the third region to the second oligonucleotide strand nucleotide of the third region that base pairs with the most 3' nucleotide of the first oligonucleotide strand of the third region. In another embodiment, the nucleotide sequence that attaches the first oligonucleotide strand of the third region and the second oligonucleotide strand of the third region has a tetraloop. In an additional embodiment, the nucleotide sequence that attaches the first oligonucleotide strand of the third region and the second oligonucleotide strand of the third region includes a hairpin, a chemical linker or an extended loop.

[0029] In one embodiment, one or both of the first and second oligonucleotide strands of any of the first, second or third regions has a 5' phosphate. In another embodiment, the nucleic acid duplex is cleaved endogenously in a mammalian cell by RNase H. In a further embodiment, the endogenous RNase H cleavage generates a nucleic acid duplex that is cleaved endogenously in the mammalian cell by Dicer. In another embodiment, the endogenous RNase H cleavage generates two nucleic acid duplexes that are each cleaved endogenously in the mammalian cell by Dicer.

[0030] In an additional embodiment, the nucleic acid duplex is cleaved endogenously in a mammalian cell by Dicer. In one embodiment, the nucleic acid duplex is cleaved endogenously in a mammalian cell to produce a double-stranded nucleic acid of 19-23 nucleotides in length that reduces target gene expression.

[0031] In another embodiment, a nucleotide of the second or first oligonucleotide strand of any of the first, second or third regions is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

[0032] In a further embodiment, the isolated nucleic acid duplex has a phosphate backbone modification that is a phosphonate, a phosphorothioate or a phosphotriester.

[0033] In one embodiment, at least 50% of the ribonucleotide residues of positions 1 to 23 of the first oligonucleotide strand that base pair with ribonucleotides of the second oligonucleotide strand to form a duplex are unmodified ribonucleotides. In another embodiment, at least 50% of all ribonucleotides of the nucleic acid duplex are unmodified ribonucleotides.

[0034] In a further embodiment, the first and second oligonucleotide strands of the third region are joined by a chemical linker. In another embodiment, the 3' terminus of the first oligonucleotide strand of the third region and the 5' terminus of the second oligonucleotide strand of the third region are joined by a chemical linker.

[0035] In one embodiment, positions 24 and greater of the first oligonucleotide strand of the first region comprise between one and 12 deoxyribonucleotide residues, where each of the deoxynucleotide residues of the first oligonucleotide strand of the first region base pairs with a deoxyribonucleotide of the second oligonucleotide strand of the first region to form a duplex.

[0036] In another embodiment, the first oligonucleotide strand of the first or third regions has a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95% or 100% complementary to the second oligonucleotide strand nucleotide sequence of the respective first or third regions.

[0037] Another aspect of the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 35 ribonucleotides in length; a second region possessing a first oligonucleotide strand and a second oligonucleotide strand having a DNA:DNA duplex having a length of DNA between two and forty base pairs, where the second region is covalently attached to the first region by an attachment that is either a covalent bond connecting the most 5' nucleotide of the first strand of the second region to the most 3' nucleotide of the first strand of the first region or a covalent bond connecting the most 3' nucleotide of the second oligonucleotide strand of the second region to the most 5' nucleotide of the second oligonucleotide strand of the first region, or both; and a third region possessing a first oligonucleotide strand having ribonucleotides and having a 3' terminus and a second oligonucleotide strand having ribonucleotides and having a 5' terminus, where the third region is covalently attached to the second region by an attachment that is either a covalent bond connecting the most 5' nucleotide of the first strand of the third region to the most 3' nucleotide of the first strand of the second region or a covalent bond connecting the most 3' nucleotide of the second oligonucleotide strand of the third region to the most 5' nucleotide of the second oligonucleotide strand of the second region, or both, where the nucleotides of the first and second oligonucleotide strands of the third region form a duplex of between 23 and 35 ribonucleotides in length.

[0038] In one embodiment, the first region includes a duplex of at least 25 nucleotides in length. In another embodiment, the first region includes a duplex of between 26 and 35 nucleotides in length. In an additional embodiment, the first region includes a duplex of between 26 and 30 nucleotides in length. In a further embodiment, the third region includes a duplex of at least 25 nucleotides in length. In another embodiment, the third region includes a duplex of between 26 and 35 nucleotides in length. In one embodiment, the third region includes a duplex of between 26 and 30 nucleotides in length.

[0039] In an additional embodiment, the second oligonucleotide strand of the first region is sufficiently complementary to a first target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target gene expression when the nucleic acid duplex is introduced into a mammalian cell. In a further embodiment, the second oligonucleotide strand of the third region is sufficiently complementary to a second target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target gene expression when the nucleic acid duplex is introduced into a mammalian cell.

[0040] In another embodiment, the first oligonucleotide strand of the third region is sufficiently complementary to a second target RNA along at least 19 nucleotides of the first oligonucleotide strand length to reduce target gene expression when the nucleic acid duplex is introduced into a mammalian cell. In an additional embodiment, the second oligonucleotide strand of the first region possesses a 3' overhang of 1-4 nucleotides in length. In another embodiment, the first oligonucleotide strand of the third region possesses a 3' overhang of 1-4 nucleotides in length. In one embodiment, the 3' overhang is 1-3 nucleotides in length. In another embodiment, the 3' overhang is 1-2 nucleotides in length.

[0041] In an additional embodiment, the nucleotides of the 3' overhang comprise a modified nucleotide. In another embodiment, the modified nucleotide of the 3' overhang is a 2'-O-methyl ribonucleotide.

[0042] In a further embodiment, all nucleotides of the 3' overhang are modified nucleotides. In an additional embodiment, the 3' overhang is two nucleotides in length and where the modified nucleotide of the 3' overhang is a 2'-O-methyl modified ribonucleotide.

[0043] In a further embodiment, the second oligonucleotide strand of the first region, starting from the nucleotide residue of the second oligonucleotide strand of the first region that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand of the first region, has unmodified nucleotide residues at all positions from position 20 to the most 5' residue of the second oligonucleotide strand of the first region. In another embodiment, the first oligonucleotide strand of the third region, starting from the nucleotide residue of the first oligonucleotide strand of the third region that is complementary to the 5' terminal nucleotide residue of the second oligonucleotide strand of the third region, has unmodified nucleotide residues at all positions from position 20 to the most 5' residue of the first oligonucleotide strand of the third region.

[0044] In one embodiment, the second oligonucleotide strand of the first region has modified nucleotides at positions 1, 2, and 3 from the 3' terminus of the second oligonucleotide strand of the first region. In an additional embodiment, the first oligonucleotide strand of the third region has modified nucleotides at positions 1, 2, and 3 from the 3' terminus of the first oligonucleotide strand of the third region. In another embodiment, at least the two most 3' nucleotide residues of the first oligonucleotide strand of the first region are deoxyribonucleotides that base pair with two deoxyribonucleotides of the second oligonucleotide strand of the first region.

[0045] In one embodiment, at least the two most 5' nucleotide residues of the first oligonucleotide strand of the third region are deoxyribonucleotides that base pair with two deoxyribonucleotides of the second oligonucleotide strand of the third region. In an additional embodiment, the two most 3' nucleotide residues of the second oligonucleotide strand of the second region are modified ribonucleotides. In a further embodiment, the two most 5' nucleotide residues of the second oligonucleotide strand of the second region are modified ribonucleotides.

[0046] In another embodiment, the second oligonucleotide strand of the third region, starting from the most 3' nucleotide residue of the second oligonucleotide strand of the third region, has alternating modified and unmodified nucleotide residues.

[0047] In one embodiment, the 3' terminus of the first oligonucleotide strand of the third region and the 5' terminus of the second oligonucleotide strand of the third region form a blunt end. In another embodiment, at least one of positions 1, 2 or 3 from the 3' terminus of the 3' terminus of the first oligonucleotide strand of the third region is a deoxyribonucleotide.

[0048] In an additional embodiment, the deoxynucleotide residues of the second region that comprise the DNA:DNA duplex are unmodified deoxyribonucleotides.

[0049] In a further embodiment, at least 50% of all deoxyribonucleotides of the nucleic acid duplex are unmodified deoxyribonucleotides. In an additional embodiment, the first oligonucleotide strand of the third region is attached to the second oligonucleotide strand of the third region by a nucleotide sequence, where the nucleotide sequence attaches the most 3' nucleotide of the first oligonucleotide strand of the third region that base pairs with a nucleotide of the second oligonucleotide strand of the third region to the second oligonucleotide strand nucleotide of the third region that base pairs with the most 3' nucleotide of the first oligonucleotide strand of the third region.

[0050] In one embodiment, the nucleotide sequence that attaches the first oligonucleotide strand of the third region and the second oligonucleotide strand of the third region has a tetraloop. In another embodiment, the nucleotide sequence that attaches the first oligonucleotide strand of the third region and the second oligonucleotide strand of the third region has a hairpin, a chemical linker or an extended loop.

[0051] In an additional embodiment, one or both of the first and second oligonucleotide strands of any of the first, second or third regions has a 5' phosphate.

[0052] In another embodiment, the nucleic acid duplex is cleaved endogenously in a mammalian cell by Dicer. In a related embodiment, the nucleic acid duplex is cleaved twice endogenously in a mammalian cell by Dicer.

[0053] In a further embodiment, the nucleic acid duplex is cleaved endogenously in a mammalian cell to produce a double-stranded nucleic acid of 19-23 nucleotides in length that reduces target gene expression. In an additional embodiment, a nucleotide of the second or first oligonucleotide strand of any of the first, second or third regions is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

[0054] In another embodiment, at least 50% of the ribonucleotide residues of positions 1 to 23 of the first oligonucleotide strand that base pair with ribonucleotides of the second oligonucleotide strand to form a duplex are unmodified ribonucleotides. In a further embodiment, at least 50% of all ribonucleotides of the nucleic acid duplex are unmodified ribonucleotides.

[0055] In an additional embodiment, the first and second oligonucleotide strands of the third region are joined by a chemical linker. In a related embodiment, the 3' terminus of the first oligonucleotide strand of the third region and the 5' terminus of the second oligonucleotide strand of the third region are joined by a chemical linker.

[0056] In an additional embodiment, positions 24 and greater of the first oligonucleotide strand of the first region comprise between one and 12 deoxyribonucleotide residues, where each of the deoxynucleotide residues of the first oligonucleotide strand of the first region base pairs with a deoxyribonucleotide of the second oligonucleotide strand of the first region to form a duplex.

[0057] In one embodiment, the first oligonucleotide strand of the first or third regions has a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95% or 100% complementary to the second oligonucleotide strand nucleotide sequence of the respective first or third regions.

[0058] In another embodiment, the isolated nucleic acid duplex is at least 50% more effective at target gene inhibition in a mammalian cell contacted with a fixed concentration of the nucleic acid duplex than a corresponding bifunctional siRNA agent (e.g., a bifunctional agent having two 19-21mer siRNAs linked via a RNase H cleavable sequence) at the same concentration. In a further embodiment, the isolated nucleic acid duplex possesses a duration of target gene inhibition in a mammalian cell contacted with a fixed concentration of the nucleic acid duplex that is at least 25% longer than a corresponding bifunctional siRNA agent at the same concentration.

[0059] A further aspect of the invention provides a method for reducing expression of a first target gene and a second target gene in a cell, by contacting a cell with an isolated nucleic acid duplex of the invention in an amount effective to reduce expression of a first target gene and a second target gene in a cell more than two unattached reference dsRNAs (optionally, 19-21mer siRNAs).

[0060] Another aspect of the invention provides a method for reducing expression of a first target gene and a second target gene in an animal, involving administering to an animal an isolated nucleic acid duplex of the invention in an amount effective to reduce expression of a first target gene and a second target gene in a cell of the animal more than two unattached reference dsRNAs.

[0061] In an additional aspect, the invention provides a pharmaceutical composition for reducing expression of a first target gene and a second target gene in a cell of a subject including an isolated nucleic acid duplex of the invention in an amount effective to reduce expression of a first target gene and a second target gene in a cell, and a pharmaceutically acceptable carrier.

[0062] Another aspect of the invention provides a pharmaceutical composition for reducing expression of a first target gene and a second target gene in a cell of a subject that includes an isolated nucleic acid duplex of the invention in an amount effective to reduce expression of a first target gene and a second target gene in a cell more than two unattached reference dsRNAs (optionally, 19-21mer siRNAs), and a pharmaceutically acceptable carrier.

[0063] A further aspect of the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; and a second region possessing a first oligonucleotide strand and a second oligonucleotide strand having a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of the second region in an RNase H cleavage assay, where the second region is covalently attached to the first region by an attachment that is either a covalent bond connecting the most 5' nucleotide of the first strand of the second region to the most 3' nucleotide of the first strand of the first region or a covalent bond connecting the most 3' nucleotide of the second oligonucleotide strand of the second region to the most 5' nucleotide of the second oligonucleotide strand of the first region, or both, where the 5' terminal residue of the second oligonucleotide strand or the 3' terminal residue of the first oligonucleotide strand of the second region is covalently attached to a functional group.

[0064] An additional aspect of the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; and a second region possessing a RNA:DNA duplex, where the RNA:DNA duplex has a first oligonucleotide strand having a 3' terminus, where the most 5' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the first region, and a second oligonucleotide strand having a 5' terminus, where the most 3' nucleotide of the second oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the second oligonucleotide strand of the first region, where the first oligonucleotide strand of the second region has between four and twenty deoxyribonucleotides that form a RNA:DNA duplex with ribonucleotides of the second oligonucleotide strand of the second region, where the 5' terminal residue of the second oligonucleotide strand of the second region is covalently attached to a functional group.

[0065] Another aspect of the invention provides an isolated nucleic acid duplex having a first region possessing a first oligonucleotide strand having ribonucleotides and having a 5' terminus and a second oligonucleotide strand having ribonucleotides and having a 3' terminus, where the nucleotides of the first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; and a second region possessing a RNA:DNA duplex, where the RNA:DNA duplex has a first oligonucleotide strand having a 3' terminus, where the most 5' nucleotide of the first oligonucleotide strand of the second region is covalently attached to the most 3' nucleotide of the first oligonucleotide strand of the first region, and a second oligonucleotide strand having a 5' terminus, where the most 3' nucleotide of the second oligonucleotide strand of the second region is covalently attached to the most 5' nucleotide of the second oligonucleotide strand of the first region, where the second oligonucleotide strand of the second region has between four and forty deoxyribonucleotides that form a RNA:DNA duplex with ribonucleotides of the first oligonucleotide strand of the second region, where the 3' terminal residue of the first oligonucleotide strand of the second region is covalently attached to a functional group.

[0066] In one embodiment, the second oligonucleotide strand of the first region is sufficiently complementary to a target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target gene expression when the nucleic acid duplex is introduced into a mammalian cell.

[0067] In another embodiment, the functional group is a ligand for a cellular receptor, a protein localization sequence, an antibody; a nucleic acid aptamer, a vitamin or other co-factor, a polymer, a phospholipid, cholesterol, a polyamine, an intercalator, a reporter molecule, a polyamine, a polyamide, polyethylene glycol, polyether, a group that enhances a pharmacodynamic property of a nucleic acid agent, a group that enhances a pharmacokinetic property of a nucleic acid agent or an active drug substance. In an additional embodiment, the functional group is attached to the second region by a linking moiety. In another embodiment, the functional group improves formulation, biodistribution, adsorption, metabolism, pharmacodynamics or cellular uptake of the nucleic acid duplex.

[0068] A further aspect of the invention provides a method for reducing expression of a target gene in a cell by contacting a cell with an isolated nucleic acid duplex of the invention in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA.

[0069] An additional aspect of the invention provides a method for reducing expression of a target gene in an animal, involving administering to an animal an isolated nucleic acid duplex of the invention in an amount effective to reduce expression of a target gene in a cell of the animal in comparison to a reference dsRNA.

[0070] In another aspect, the invention provides a pharmaceutical composition for reducing expression of a target gene in a cell of a subject that includes an isolated nucleic acid duplex of the invention in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA, and a pharmaceutically acceptable carrier.

[0071] In an additional aspect, the invention provides a method of synthesizing an isolated nucleic acid duplex of the invention by chemically or enzymatically synthesizing the nucleic acid duplex.

[0072] In a further aspect, the invention provides a kit having an isolated nucleic acid duplex of the invention, with instructions for its use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1 shows the structure of an exemplary bifunctional DsiRNA agent of the invention, with the two DsiRNA agents that may be liberated via RNase H cleavage oriented in tandem within the uncleaved bifunctional DsiRNA agent shown.

[0074] FIG. 2 shows the structure of an exemplary bifunctional DsiRNA agent of the invention, with the two DsiRNA agents that may be liberated via RNase H cleavage oriented in opposite directions to one another within the uncleaved bifunctional DsiRNA agent shown.

[0075] FIG. 3 shows the structure of an exemplary RNase H-cleavable agent designed to release both a DsiRNA agent and a functional group upon RNase H cleavage.

[0076] FIG. 4 shows a bifunctional DsiRNA agent having two DsiRNA agents directed to independent RNA targets that are joined by a tract of double stranded DNA. Such agents are not reliant upon RNase H for cleavage, but instead rely upon Dicer cleavage to produce effective inhibitory products.

DETAILED DESCRIPTION

[0077] The invention provides compositions and methods for reducing expression of one or more target genes in a cell, involving contacting a cell with an isolated double stranded nucleic acid double stranded nucleic acid in an amount effective to reduce expression of one or more target genes in a cell. In certain embodiments, the double stranded nucleic acids of the invention comprise RNase H-cleavable double stranded nucleic acid regions, which are employed to attach DsiRNA agents to one another within one compound (precursor) agent (termed a "bifunctional DsiRNA" herein). Alternatively, such RNase H-cleavable double stranded nucleic acid double stranded nucleic acid regions are used to attach a DsiRNA agent to a functional group (e.g., a functional group of any type suitable to improve a desired property of DsiRNA in vivo, such as improvement of formulation, biodistribution, adsorption, metabolism, pharmacodynamics, cellular uptake, etc.; such agents are also referred to herein as "functional group-tethered DsiRNA agents"). In additional embodiments, DsiRNA moieties can also be joined by a double stranded DNA joining sequence, which allows for construction of another form of bifunctional DsiRNA of the instant invention that is not reliant upon/does not provoke RNase H-mediated cleavage.

DEFINITIONS

[0078] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0079] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0080] As used herein, "nucleotide" is used as recognized in the art to include those with 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, e.g., 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, Nucleic Acids Res. 22:2183, 1994. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, hypoxanthine, 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., Biochemistry 35:14090, 1996; 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.

[0081] As used herein, a "double-stranded nucleic acid" is a molecule comprising two oligonucleotide strands which form a duplex. A double stranded nucleic acid may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. The double-stranded NAs of the instant invention are substrates for proteins and protein complexes in the RNA interference pathway, e.g., Dicer and RISC. Structures of double stranded nucleic acids of the invention are shown in FIG. 1, and characteristically comprise an RNA duplex in a region that is capable of functioning as a Dicer substrate siRNA (DsiRNA) and a DNA duplex comprising at least one deoxyribonucleotide, which is located at a position 3' of the projected Dicer cleavage site of the first strand of the DsiRNA/DNA agent, base paired with a cognate deoxyribonucleotide of the second strand, which is located at a position 5' of the projected Dicer cleavage site of the second strand of the DsiRNA/DNA agent.

[0082] As used herein, "duplex" refers to a double helical structure formed by the interaction of two single stranded nucleic acids. According to the present invention, a duplex may contain first and second strands which are sense and antisense, or which are target and antisense. A duplex is typically formed by the pairwise hydrogen bonding of bases, i.e., "base pairing", between two single stranded nucleic acids which are oriented antiparallel with respect to each other. Base pairing in duplexes generally occurs by Watson-Crick base pairing, e.g., guanine (G) forms a base pair with cytosine (C) in DNA and RNA (thus, the cognate nucleotide of a guanine deoxyribonucleotide is a cytosine deoxyribonucleotide, and vice versa), adenine (A) forms a base pair with thymine (T) in DNA, and adenine (A) forms a base pair with uracil (U) in RNA. Conditions under which base pairs can form include physiological or biologically relevant conditions (e.g., intracellular: pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion). Furthermore, duplexes are stabilized by stacking interactions between adjacent nucleotides. As used herein, a duplex may be established or maintained by base pairing or by stacking interactions. A duplex is formed by two complementary nucleic acid strands, which may be substantially complementary or fully complementary (see below).

[0083] By "complementary" or "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or Hoogsteen base pairing. In reference to the nucleic acid molecules of the present disclosure, 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., CSH Symp. Quant. Biol. LII, pp. 123-133, 1987; Frier, et al., Proc. Nat. Acad. Sci. USA 83:9373-9377, 1986; Turner, et al., J. Am. Chem. Soc. 109:3783-3785, 1987). 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). To determine that a percent complementarity is of at least a certain percentage, 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 is calculated and rounded to the nearest whole number (e.g., 12, 13, 14, 15, 16, or 17 nucleotides out of a total of 23 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 23 nucleotides represents 52%, 57%, 61%, 65%, 70%, and 74%, respectively; and has at least 50%, 50%, 60%, 60%, 70%, and 70% complementarity, respectively). As used herein, "substantially complementary" refers to complementarity between the strands such that they are capable of hybridizing under biological conditions. Substantially complementary sequences have 60%, 70%, 80%, 90%, 95%, or even 100% complementarity. Additionally, techniques to determine if two strands are capable of hybridizing under biological conditions by examining their nucleotide sequences are well known in the art.

[0084] Single-stranded nucleic acids that base pair over a number of bases are said to "hybridize." Hybridization is typically determined under physiological or biologically relevant conditions (e.g., intracellular: pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion). Hybridization conditions generally contain a monovalent cation and biologically acceptable buffer and may or may not contain a divalent cation, complex anions, e.g. gluconate from potassium gluconate, uncharged species such as sucrose, and inert polymers to reduce the activity of water in the sample, e.g. PEG. Such conditions include conditions under which base pairs can form.

[0085] Hybridization is measured by the temperature required to dissociate single stranded nucleic acids forming a duplex, i.e., (the melting temperature; Tm). Hybridization conditions are also conditions under which base pairs can form. Various conditions of stringency can be used to determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). Stringent temperature conditions will ordinarily include temperatures of at least about 30.degree. C., more preferably of at least about 37.degree. C., and most preferably of at least about 42.degree. C. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10.degree. C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(.degree. C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(.degree. C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1.times.SSC=0.165 M). For example, a hybridization determination buffer is shown in Table 1.

TABLE-US-00001 TABLE 1 To make 50 final conc. Vender Cat# Lot# m.w./Stock ml solution NaCl 100 mM Sigma S-5150 41K8934 5M 1 mL KCl 80 mM Sigma P-9541 70K0002 74.55 0.298 g MgCl.sub.2 8 mM Sigma M-1028 120K8933 1M 0.4 mL sucrose 2% w/v Fisher BP220-212 907105 342.3 1 g Tris-HCl 16 mM Fisher BP1757-500 12419 1M 0.8 mL NaH.sub.2PO.sub.4 1 mM Sigma S-3193 52H-029515 120.0 0.006 g EDTA 0.02 mM Sigma E-7889 110K89271 0.5M 2 .mu.L H.sub.2O Sigma W4502 51K2359 to 50 mL pH = 7.0 adjust with at 20.degree. C. HCl

[0086] Useful variations on hybridization conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Antisense to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0087] As used herein, "oligonucleotide strand" is a single stranded nucleic acid molecule. An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2' modifications, synthetic base analogs, etc.) or combinations thereof. Such modified oligonucleotides can be preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.

[0088] Certain nucleic acid duplex agents of this invention are chimeric double stranded nucleic acids. "Chimeric double stranded nucleic acids" or "chimeras", in the context of this invention, are double stranded nucleic acids which contain two or more chemically distinct regions, each made up of at least one nucleotide. These double stranded nucleic acids typically contain at least one region primarily comprising ribonucleotides (optionally including modified ribonucleotides) that form a Dicer substrate siRNA ("DsiRNA") molecule. This DsiRNA region is covalently attached (at one or both strands) to a second region comprising a RNA:DNA duplex that forms an RNase H substrate. This RNA:DNA duplex region is, in turn, covalently attached to a third region comprising a DsiRNA moiety, or is covalently attached to a moiety comprising a functional group. Any of the above-described chimeric double stranded nucleic acid regions may also include modified or synthetic nucleotides and/or modified or synthetic deoxyribonucleotides.

[0089] As used herein, the term "ribonucleotide" encompasses natural and synthetic, unmodified and modified ribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between ribonucleotides in the oligonucleotide. As used herein, the term "ribonucleotide" specifically excludes a deoxyribonucleotide, which is a nucleotide possessing a single proton group at the 2' ribose ring position.

[0090] As used herein, the term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between deoxyribonucleotide in the oligonucleotide.

[0091] As used herein, the term "RNAse H" refers to an enzyme that cleaves RNA that is part of a RNA:DNA heteroduplex. Incorporation of one or more DNA residues within a first strand of a duplex RNA agent allows the oligoribonucleotide region of a second strand, within the oligoribonucleotide region that anneals to the one or more DNA residues of the first strand to be cleaved at the hybridized RNA residues. In certain non-mammalian animals, some RNAse H enzymes require only one ribonucleotide in an oligonucleotide as substrate. However, mammalian RNase H enzymes require a segment of at lease four ribonucleotides. RNAse H activity can be found in some polymerases, including reverse transcriptase. RNAse H can also be a separate enzyme. Suitable RNAse H enzymes include human and E. coli RNAse Hs. The human RNase H family includes the following: RNase H1 (GenBank Accession No. NM.sub.--002936.3); RNase H2A (GenBank Accession No. NM.sub.--006397.2); RNase H.sub.2B (GenBank Accession No. NM.sub.--024570.1); and RNase H.sub.2C (GenBank Accession No. NM.sub.--032193.3). RNase H activity is also observed in human EXO1 (GenBank Accession Nos. NM.sub.--130398.2, NM.sub.--006027.3 and NM.sub.--003686.3 isoforms). An additional RNAse H that can be used is Thermus thermophilus, or Tth, RNAse H.

[0092] RNase H cleavage of an agent of the instant invention can be assessed by any art-recognized method. Exemplary methods for detecting RNase H cleavage of a candidate substrate (e.g., where the RNase H substrate is a DsiRNA agent joined to a region comprising a DNA:RNA duplex sequence) include, e.g., detection of RNase H cleavage product(s) based upon the appearance of appropriately-sized bands on a gel (e.g., gel electrophoresis/electrophoretic mobility test and, if necessary, associated nucleic acid hybridization techniques known in the art). In addition, RNase H cleavage products also can be assessed via performance of mass spectroscopy (e.g., time of flight) upon a candidate substrate solution that has been exposed to RNase H (e.g., cleavage products derived from mammalian cell culture or lysate, and/or cleavage products found in an appropriate in vitro assay for RNase H cleavage.

[0093] In certain embodiments, the detectability of an RNase H cleavage product in a solution is assessed. The lower limit at which an RNase H cleavage product becomes detectable is likely to depend upon the nature of the RNase H cleavage assay employed (e.g., mass spectroscopy approaches will tend to detect cleavage product at a lower threshold than gel electrophoresis methods). The skilled artisan will recognize how to set appropriate limits in performing such assays to classify a product as detectable or not detectable. In certain embodiments, e.g., a cleavage product is considered detectable if at least 1% of input agent is cleaved in a given time (e.g., 30 minute treatment) at a given concentration of RNase H enzyme (e.g., 1 unit/mL). In related embodiments, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or even at least 90% of input agent must yield an appropriate cleavage product for the product to be considered detectable or for the input agent to be classified as an RNase H cleavable agent.

[0094] RNase H hydrolyzes RNA in RNA-DNA hybrids. This enzyme was first identified in calf thymus but has subsequently been described in a variety of organisms (Stein, H. and Hausen, P., Science, 1969, 166, 393-395; Hausen, P. and Stein, H., Eur. J. Biochem., 1970, 14, 278-283). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (Itaya, M. and Kondo K. Nucleic Acids Res., 1991, 19, 4443-4449; Itaya et al., Mol. Gen. Genet., 1991 227, 438-445; Kanaya, S., and Itaya, M., J. Biol. Chem., 1992, 267, 10184-10192; Busen, W., J. Biol. Chem., 1980, 255, 9434-9443; Rong, Y. W. and Carl, P. L., 1990, Biochemistry 29, 383-389; Eder et al., Biochimie, 1993 75, 123-126). Although RNase Hs constitute a family of proteins of varying molecular weight, nucleolytic activity and substrate requirements appear to be similar for the various isotypes. For example, all RNase Hs studied to date function as endonucleases, exhibiting limited sequence specificity and requiring divalent cations (e.g., Mg.sup.2+, Mn.sup.2+) to produce cleavage products with 5' phosphate and 3' hydroxyl termini (Crouch, R. J., and Dirksen, M. L., Nuclease, Linn, S, M., & Roberts, R. J., Eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y. 1982, 211-241).

[0095] To evaluate the binding affinity (and specificity) of a human RNase H1 for a substrate, a competitive cleavage assay in which increasing concentrations of noncleavable substrates are added can also be used. Using this approach, the Ki is formally equivalent to the Kd for the competing substrates. Such assays have been described in greater detail, e.g., in U.S. Application No. 2007/0292875.

[0096] As used herein, the term "functional group" refers to a moiety that improves any desired property of a DsiRNA-containing agent in vivo. Such properties include, but are not limited to, improvement of formulation, biodistribution, adsorption, metabolism, pharmacodynamics, and cellular uptake. In certain embodiments, such a "functional group" is selected from the following: ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; polyamines, such as spermine or spermidine; intercalators; reporter molecules; polyamines; polyamides; polyethylene glycols; polyethers; groups that enhance the pharmaco-dynamic properties of nucleic acid agents, such as cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins and dyes; groups that enhance the pharmacodynamic properties, such as groups that improve uptake, enhance resistance to degradation, enhance RISC residency and/or strengthen sequence-specific hybridization with the target nucleic acid; groups that enhance the pharmacokinetic properties, such as groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Functional group moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Nucleic acid agents of the invention may also be attached to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

[0097] In certain embodiments, a nucleic acid duplex of the invention comprises at least one duplex region of at least 23 nucleotides in length, within which at least 50% of all nucleotides are unmodified ribonucleotides. As used herein, the term "unmodified ribonucleotide" refers to a ribonucleotide possessing a hydroxyl (--OH) group at the 2' position of the ribose sugar.

[0098] In certain embodiments, a nucleic acid duplex of the invention comprises at least one region, located 3' of the projected Dicer cleavage site on the first strand and 5' of the projected Dicer cleavage site on the second strand, having a length of at least 4 base paired nucleotides that form an RNA:DNA duplex, wherein at least 50% of all deoxynucleotides within this region of at least 4 base paired nucleotides in length are unmodified deoxyribonucleotides. As used herein, the term "unmodified deoxyribonucleotide" refers to a ribonucleotide possessing a single proton at the 2' position of the ribose sugar.

[0099] As used herein, "antisense strand" refers to a single stranded nucleic acid molecule which has a sequence complementary to that of a target RNA. When the antisense strand contains modified nucleotides with base analogs, it is not necessarily complementary over its entire length, but must at least duplex with a target RNA.

[0100] As used herein, "sense strand" refers to a single stranded nucleic acid molecule which has a sequence complementary to that of an antisense strand. When the antisense strand contains modified nucleotides with base analogs, the sense strand need not be complementary over the entire length of the antisense strand, but must at least duplex with the antisense strand.

[0101] As used herein, "guide strand" refers to a single stranded nucleic acid molecule of a dsRNA, which has a sequence complementary to that of a target RNA, and results in RNA interference by binding to a target RNA. After cleavage of the dsRNA by Dicer, a fragment of the guide strand remains associated with RISC, binds a target RNA as a component of the RISC complex, and promotes cleavage of a target RNA by RISC. As used herein, the guide strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity (e.g., a "nick" referring to the absence of solely a single phosphodiester bond between adjacent nucleotides, a "gap" referring to the absence of at least one internal nucleotide from a length of duplex sequence), preferably at a site that is cleaved by Dicer. A guide strand is an antisense strand.

[0102] As used herein, "target RNA" refers to an RNA that would be subject to modulation guided by the antisense strand, such as targeted cleavage or steric blockage. The target RNA could be, for example genomic viral RNA, mRNA, a pre-mRNA, or a non-coding RNA. The preferred target is mRNA, such as the mRNA encoding a disease associated protein, such as ApoB, Bcl2, Hif-1alpha, Survivin or a p21 ras, such as Ha. ras, K-ras or N-ras.

[0103] As used herein, "passenger strand" refers to an oligonucleotide strand of a dsRNA, which has a sequence that is complementary to that of the guide strand. As used herein, the passenger strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity (e.g., a "nick" referring to the absence of solely a single phosphodiester bond between adjacent nucleotides, a "gap" referring to the absence of at least one internal nucleotide from a length of duplex sequence), preferably at a site that is cleaved by Dicer. A passenger strand is a sense strand.

[0104] As used herein, "Dicer" refers to an endoribonuclease in the RNase III family that cleaves a dsRNA, e.g., double-stranded RNA (dsRNA) or pre-microRNA (miRNA), into double-stranded nucleic acid fragments about 20-25 nucleotides long, usually with a two-base overhang on the 3' end. With respect to the dsRNAs of the invention, the duplex formed by a dsRNA is recognized by Dicer and is a Dicer substrate on at least one strand of the duplex. Dicer catalyzes the first step in the RNA interference pathway, which consequently results in the degradation of a target RNA. The protein sequence of human Dicer is provided at the NCBI database under accession number NP.sub.--085124, hereby incorporated by reference.

[0105] Dicer "cleavage" is determined as follows (e.g., see Collingwood et al., Oligonucleotides 18:187-200 (2008)). In a Dicer cleavage assay, RNA duplexes (100 .mu.mol) are incubated in 20 .mu.L of 20 mM Tris pH 8.0, 200 mM NaCl, 2.5 mM MgCl2 with or without 1 unit of recombinant human Dicer (Genlantis, San Diego, Calif.) at 37.degree. C. for 18-24 hours. Samples are desalted using a Performa SR 96-well plate (Edge Biosystems, Gaithersburg, Md.). Electrospray-ionization liquid chromatography mass spectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment with Dicer is done using an Oligo HTCS system (Novatia, Princeton, N.J.; Hail et al., 2004), which consists of a ThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processing software and Paradigm MS4 HPLC (Michrom BioResources, Auburn, Calif.). In this assay, Dicer cleavage occurs where at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% of the Dicer substrate dsRNA, (i.e., 25-30 bp, dsRNA, preferably 26-30 by dsRNA) is cleaved to a shorter dsRNA (e.g., 19-23 by dsRNA, preferably, 21-23 by dsRNA). The orientation of Dicer cleavage may also be determined in such an assay, or can be determined via evaluation of the functional efficacy of resultant RNAi agents post-Dicer cleavage. In a scenario in which Dicer exhibited no end preference, the orientation of Dicer cleavage of a given agent would be split equally between the two orientations of a given double-stranded nucleic acid, e.g., 50% of molecules releasing one orientation of cleavage product, while 50% of resultant molecules show the opposite orientation. However, in view of art-recognized end structures believed to orient Dicer cleavage, one orientation of DsiRNA cleavage may be favored over another, resulting in, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, etc. of Dicer-cleaved products resulting from a given DsiRNA being the cleavage product of a single preferred orientation of DsiRNA agent, to the corresponding reduction or exclusion of the cleavage product that would result/results from Dicer cleavage of the input DsiRNA agent in the opposite orientation. Thus, the orientation of Dicer cleavage of a given agent can be tested using a functional (e.g., target RNA inhibition measurement) and/or marker- or label-based readout, or can be assessed using any other art-recognized means of such detection (e.g., mass spectroscopy may also be used to assess the identity of Dicer cleavage products).

[0106] As used herein, "Dicer cleavage site" refers to the sites at which Dicer cleaves a dsRNA (e.g., the dsRNA region of a double stranded nucleic acid of the invention). Dicer contains two RNase III domains which typically cleave both the sense and antisense strands of a dsRNA. The average distance between the RNase III domains and the PAZ domain determines the length of the short double-stranded nucleic acid fragments it produces and this distance can vary (Macrae I, et al. (2006). "Structural basis for double-stranded RNA processing by Dicer". Science 311 (5758): 195-8.). For the RNase H-cleavable double stranded nucleic acid agents of the invention, the most prominent Dicer cleavage site of the first DsiRNA agent is generally positioned about 21 nucleotides from the 5' terminus of the first strand (though certain modifications of the double stranded nucleic acid are capable of shifting the location and distribution of Dicer cleavage products within such first region DsiRNA agent). While dependent upon the length of this first region DsiRNA, DNA:RNA hybrid sequences of the double stranded nucleic acid agents of the invention generally commence at a position between about position 24 and 36 of the first strand (where position 1 is the 5' terminal residue of the first strand).

[0107] As used herein, "overhang" refers to unpaired nucleotides, in the context of a duplex having two or four free ends at either the 5' terminus or 3' terminus of a dsRNA. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand.

[0108] As used herein, "target" refers to any nucleic acid sequence whose expression or activity is to be modulated. In particular embodiments, the target refers to an RNA which duplexes to a single stranded nucleic acid that is an antisense strand in a RISC complex. Hybridization of the target RNA to the antisense strand results in processing by the RISC complex. Consequently, expression of the RNA or proteins encoded by the RNA, e.g., mRNA, is reduced.

[0109] As used herein, "reference" is meant a standard or control. As is apparent to one skilled in the art, an appropriate reference is where only one element is changed in order to determine the effect of the one element.

[0110] By the term "antisense agent" is meant a polynucleotide fragment (comprising either deoxyribonucleotides, ribonucleotides, synthetic nucleotides or a mixture thereof) having inhibitory antisense activity, said activity causing a decrease in the expression of the endogenous genomic copy of the corresponding gene. The sequence of the antisense agent is designed to complement a target mRNA of interest and form an RNA:antisense agent duplex. This duplex formation can prevent processing, splicing, transport or translation of the relevant mRNA. Moreover, certain antisense agents can elicit cellular RNase H activity when hybridized with the target mRNA, resulting in mRNA degradation (Calabretta et al, 1996: Antisense strategies in the treatment of leukemias. Semin Oncol. 23(1):78-87). In that case, RNase H will cleave the RNA component of the duplex and can potentially release the antisense agent to further hybridize with additional molecules of the target RNA. An additional mode of action results from the interaction of an antisense agent with genomic DNA to form a triple helix, which can be transcriptionally inactive.

[0111] As used herein, "modified nucleotide" refers to a nucleotide that has one or more modifications to the nucleoside, the nucleobase, pentose ring, or phosphate group. For example, modified nucleotides exclude ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include those naturally occurring that result from modification by enzymes that modify nucleotides, such as methyltransferases. Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications, e.g., 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH.sub.2--O-2'-bridge, 4'-(CH.sub.2).sub.2--O-2'-bridge, 2'-LNA, and 2'-O--(N-methylcarbamate) or those comprising base analogs. In connection with 2'-modified nucleotides as described for the present disclosure, by "amino" is meant 2'--NH.sub.2 or 2'-O--NH.sub.2, which can be modified or unmodified. Such modified groups are described, e.g., in Eckstein, et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic, et al., U.S. Pat. No. 6,248,878.

[0112] In reference to the nucleic acid molecules of the present disclosure, the modifications may exist in patterns on a strand of the double stranded nucleic acid. As used herein, "alternating positions" refers to a pattern where every other nucleotide is a modified nucleotide or there is an unmodified nucleotide (e.g., an unmodified ribonucleotide) between every modified nucleotide over a defined length of a strand of the dsRNA (e.g., 5'-MNMNMN-3'; 3'-MNMNMN-5'; where M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to any of the position numbering conventions described herein. The pattern of modified nucleotides at alternating positions may run the full length of the strand, but preferably includes at least 4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7 modified nucleotides, respectively. As used herein, "alternating pairs of positions" refers to a pattern where two consecutive modified nucleotides are separated by two consecutive unmodified nucleotides over a defined length of a strand of the dsRNA (e.g., 5'-MMNNMMNNMMNN-3'; 3'-MMNNMMNNMMNN-5'; where M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to any of the position numbering conventions described herein. The pattern of modified nucleotides at alternating positions may run the full length of the strand, but preferably includes at least 8, 12, 16, 20, 24, 28 nucleotides containing at least 4, 6, 8, 10, 12 or 14 modified nucleotides, respectively.

[0113] As used herein, "base analog" refers to a heterocyclic moiety which is located at the 1' position of a nucleotide sugar moiety in a modified nucleotide that can be incorporated into a nucleic acid duplex (or the equivalent position in a nucleotide sugar moiety substitution that can be incorporated into a nucleic acid duplex). In the double stranded nucleic acids of the invention, a base analog is generally either a purine or pyrimidine base excluding the common bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Base analogs can duplex with other bases or base analogs in dsRNAs. Base analogs include those useful in the compounds and methods of the invention., e.g., those disclosed in U.S. Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent Publication No. 20080213891 to Manoharan, which are herein incorporated by reference. Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 3.beta.-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3-.beta.-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-d- ione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), 1-.beta.-D-ribofuranosyl-(5-nitroindole), 1-.beta.-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S),2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer et al., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic Acids Research, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc., 119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121:2323-2324 (1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Morales et al., J. Am. Chem. Soc., 122(6):1001-1007 (2000); McMinn et al., J. Am. Chem. Soc., 121:11585-11586 (1999); Guckian et al., J. Org. Chem., 63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci., 94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin Trans., 1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1: 1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632 (2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri et al., J. Am. Chem. Soc., 117:10434-10442 (1995); and U.S. Pat. No. 6,218,108.). Base analogs may also be a universal base.

[0114] As used herein, "universal base" refers to a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a nucleic acid duplex, can be positioned opposite more than one type of base without altering the double helical structure (e.g., the structure of the phosphate backbone). Additionally, the universal base does not destroy the ability of the single stranded nucleic acid in which it resides to duplex to a target nucleic acid. The ability of a single stranded nucleic acid containing a universal base to duplex a target nucleic can be assayed by methods apparent to one in the art (e.g., UV absorbance, circular dichroism, gel shift, single stranded nuclease sensitivity, etc.). Additionally, conditions under which duplex formation is observed may be varied to determine duplex stability or formation, e.g., temperature, as melting temperature (Tm) correlates with the stability of nucleic acid duplexes. Compared to a reference single stranded nucleic acid that is exactly complementary to a target nucleic acid, the single stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid. However, compared to a reference single stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid having the mismatched base.

[0115] Some universal bases are capable of base pairing by forming hydrogen bonds between the universal base and all of the bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) under base pair forming conditions. A universal base is not a base that forms a base pair with only one single complementary base. In a duplex, a universal base may form no hydrogen bonds, one hydrogen bond, or more than one hydrogen bond with each of G, C, A, T, and U opposite to it on the opposite strand of a duplex. Preferably, the universal bases does not interact with the base opposite to it on the opposite strand of a duplex. In a duplex, base pairing between a universal base occurs without altering the double helical structure of the phosphate backbone. A universal base may also interact with bases in adjacent nucleotides on the same nucleic acid strand by stacking interactions. Such stacking interactions stabilize the duplex, especially in situations where the universal base does not form any hydrogen bonds with the base positioned opposite to it on the opposite strand of the duplex. Non-limiting examples of universal-binding nucleotides include inosine, 1-.beta.-D-ribofuranosyl-5-nitroindole, and/or 1-.beta.-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct. 11; 22(20):4039-43).

[0116] As used herein, "increase" or "enhance" is meant to alter positively by at least 5% compared to a reference in an assay. An alteration may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100% compared to a reference in an assay. By "enhance Dicer cleavage," it is meant that the processing of a quantity of a dsRNA molecule by Dicer results in more Dicer cleaved dsRNA products or that Dicer cleavage reaction occurs more quickly compared to the processing of the same quantity of a reference dsRNA in an in vivo or in vitro assay of this disclosure. In one embodiment, enhanced or increased Dicer cleavage of a dsRNA molecule is above the level of that observed with an appropriate reference dsRNA molecule. In another embodiment, enhanced or increased Dicer cleavage of a dsRNA molecule is above the level of that observed with an inactive or attenuated molecule.

[0117] As used herein "reduce" is meant to alter negatively by at least 5% compared to a reference in an assay. An alteration may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100% compared to a reference in an assay. By "reduce expression," 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 level or activity of one or more proteins or protein subunits encoded by a target gene, is reduced below that observed in the absence of the nucleic acid molecules (e.g., dsRNA molecule) in an in vivo or in vitro assay of this disclosure. In one embodiment, inhibition, down-regulation or reduction with a dsRNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with dsRNA molecules is below that level observed in the presence of, e.g., an dsRNA molecules 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 disclosure is greater in the presence of the nucleic acid molecule than in its absence.

[0118] As used herein, "cell" is meant to include both prokaryotic (e.g., bacterial) and eukaryotic (e.g., mammalian or plant) cells. Cells may be of somatic or germ line origin, may be totipotent or pluripotent, and may be dividing or non-dividing. Cells can also be derived from or can comprise a gamete or an embryo, a stem cell, or a fully differentiated cell. Thus, the term "cell" is meant to retain its usual biological meaning and can be present in any organism such as, for example, a bird, a plant, and a mammal, including, for example, a human, a cow, a sheep, an ape, a monkey, a pig, a dog, and a cat. Within certain aspects, the term "cell" refers specifically to mammalian cells, such as human cells, that contain one or more isolated dsRNA molecules of the present disclosure. In particular aspects, a cell processes dsRNAs resulting in RNA interference of target nucleic acids, and contains proteins and protein complexes required for RNAi, e.g., Dicer and RISC.

[0119] As used herein, "animal" is meant a multicellular, eukaryotic organism, including a mammal, particularly a human. The methods of the invention in general comprise administration of an effective amount of the agents herein, such as an agent of the structures of formulae herein, to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, or a symptom thereof.

[0120] By "pharmaceutically acceptable carrier" is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant disclosure in the physical location most suitable for their desired activity.

[0121] The present invention is directed to compositions that comprise both one or more double stranded RNA ("dsRNA") duplex region(s) and a RNA:DNA duplex within the same agent, and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. One of the strands of the one or more dsRNA region(s) contains a region of nucleotide sequence that has a length that ranges from about 15 to about 22 nucleotides that can direct the destruction of the RNA transcribed from a target gene. In certain embodiments, the RNA:DNA duplex region of such an agent is not complemenatary to the target RNA, and, therefore, does not enhance target RNA hybridization of the region of nucleotide sequence capable of directing destruction of a target RNA. In certain embodiments, nucleic acid duplex agents of the invention can possess strands that are chemically linked, or can also possess an extended loop, optionally comprising a tetraloop, that links the first and second strands. In some embodiments, the extended loop containing the tetraloop is at the 3' terminus of the sense strand, at the 5' terminus of the antisense strand, or both.

[0122] The nucleic acid duplex (DsiRNA/RNA:DNA duplex) agents of the instant invention can enhance the following attributes of such agents relative to DsiRNAs lacking such RNA:DNA duplex regions: potency or efficacy of compound therapeutics (specifically, co-delivery of two or more DsiRNA agents directed against the same target RNA sequence, distinct sequences within the same target RNA sequence, or two distinct target RNA sequences, in a single agent), pharmacokinetics, pharmacodynamics, intracellular uptake, nuclease stabilization, and reduced toxicity.

[0123] As used herein, the term "pharmacokinetics" refers to the process by which a drug is absorbed, distributed, metabolized, and eliminated by the body. In certain embodiments of the instant invention, enhanced pharmacokinetics of a DsiRNA/dsDNA agent relative to an appropriate control DsiRNA refers to increased absorption and/or distribution of such an agent, and/or slowed metabolism and/or elimination of such a DsiRNA/dsDNA agent from a subject administered such an agent.

[0124] As used herein, the term "pharmacodynamics" refers to the action or effect of a drug on a living organism. In certain embodiments of the instant invention, enhanced pharmacodynamics of a nucleic acid duplex agent of the invention relative to an appropriate control DsiRNA refers to an increased (e.g., more potent or more prolonged) action or effect of a nucleic acid duplex agent of the invention upon a subject administered such agent, relative to an appropriate control DsiRNA.

[0125] As used herein, the term "stabilization" refers to a state of enhanced persistence of an agent in a selected environment (e.g., in a cell or organism). In certain embodiments, the nucleic acid duplex agents of the instant invention exhibit enhanced stability relative to appropriate control DsiRNAs. Such enhanced stability can be achieved via enhanced resistance of such agents to degrading enzymes (e.g., nucleases) or other agents.

DsiRNA Design/Synthesis

[0126] It was previously shown that longer dsRNA species of from 25 to about 30 nucleotides (DsiRNAs) yield unexpectedly effective RNA inhibitory results in terms of potency and duration of action, as compared to 19-23mer siRNA agents. Without wishing to be bound by the underlying theory of the dsRNA processing mechanism, it is thought that the longer dsRNA species serve as a substrate for the Dicer enzyme in the cytoplasm of a cell. In addition to cleaving the double stranded nucleic acid of the invention into shorter segments, Dicer is thought to facilitate the incorporation of a single-stranded cleavage product derived from the cleaved double stranded nucleic acid into the RISC complex that is responsible for the destruction of the cytoplasmic RNA of or derived from the target gene. Prior studies (Rossi et al., U.S. Patent Application No. 2007/0265220) have shown that the cleavability of a dsRNA species (specifically, a DsiRNA agent) by Dicer corresponds with increased potency and duration of action of the dsRNA species. The instant invention, at least in part, provides for design of compound RNA inhibitory agents that are joined by RNase H-cleavable double-stranded nucleic acid double stranded nucleic acid sequences, such that active DsiRNA moieties and, optionally, released functional groups or payloads, are produced following exposure of such agents to an RNase H-containing environment (e.g., administration to a subject, target cell or RNase-containing solution).

[0127] Exemplary bifunctional DsiRNA structures and RNase H- and Dicer-mediated processing of such structures is shown in FIGS. 1 and 2. An exemplary structure and RNase H- and Dicer-mediated processing of a DsiRNA joined via an RNase H-cleavable double stranded nucleic acid to a functional group is shown in FIG. 3.

[0128] As depicted in FIG. 1, a double-stranded oligonucleotide is synthesized that possesses the following structure: (1) a first region having a RISC-activating domain (preferably dsRNA) of about 23 to 33 duplexed nucleotides in length (the agent shown in FIG. 1 specifically possesses a 23 nucleotide length of duplexed dsRNAs); (2) a second region that includes an RNA:DNA hybrid domain of at least four duplexed nucleotides in length, with such RNA:DNA hybrid domain constituting an RNase H-cleavable site (the agent shown in FIG. 1 specifically possesses an RNA:DNA hybrid region of 8 duplexed nucleotides in length, with only the middle four base pairs of such eight duplexed nucleotides constituting RNA:DNA base paired nucleotides wherein the ribonucleotides are unmodified ribonucleotides (the deoxyribonucleotides of this region may be either modified or unmodified deoxyribonucleotides)--indeed, modification of the ribonucleotides flanking this core, four base pair long tract of (unmodified RNA):DNA duplexed nucleotides is predicted to direct RNase H cleavage away from such modified ribonucleotide sites, instead directing such RNase H cleavage to occur at a location(s) within the (core four base pair length) (unmodified RNA):DNA duplexed nucleotides); and (3) a third region having a RISC-activating domain (preferably dsRNA) of about 23 to 33 duplexed nucleotides in length (the agent shown in FIG. 1 specifically possesses a 23 nucleotide length of duplexed dsRNAs and a two base pair length DNA:RNA duplex at the 3' terminus of the first strand/5' terminus of the second strand).

[0129] Like previously described DsiRNA agents, the first region of an RNase H-cleavable agent such as the one depicted in FIG. 1 can optionally comprise an overhang (optionally a 3' overhang of 1-4 nucleotides in length). In certain embodiments, the nucleotides of such an overhang are modified ribonucleotides, or may comprise deoxyribonucleotides. In addition, the duplexed nucleotides of the first region can also comprise modified nucleotides, e.g., modified ribonucleotides, e.g., at alternating locations within the span of the second strand that is complementary to a target RNA and that is modeled to lie 3' of the Dicer cleavage site of the DsiRNA agent that is predicted to be formed from the first and second regions via RNase H cleavage of the compound (precursor) RNase H-cleavable DsiRNA-containing starting agent. Indeed, the RNase H-processed products of the bifunctional agent shown in FIG. 1 are, in turn, processed by Dicer to yield two independent, active siRNA agents.

[0130] In view of the third region of the above-described RNase H-cleavable "bifunctional DsiRNA" also comprising a RISC-activating domain that is liberated, optionally in concert with a portion of the second region to form a DsiRNA agent, via RNase H cleavage, the nucleotides of this third region may also comprise modified nucleotides, e.g., modified ribonucleotides, e.g., at alternating locations within the span of the second strand that is complementary to a target RNA and that is modeled to lie 3' on the second strand from the Dicer cleavage site of the DsiRNA agent that is predicted to be formed from the second and third regions via RNase H cleavage of the compound (precursor) RNase H-cleavable DsiRNA-containing starting agent (bifunctional DsiRNA agent). It is noted that the first strand of this compound bifunctional DsiRNA agent depicted in FIG. 1 also possesses a discontinuity (also known as a "nick", with the first strand of this molecule optionally referred to as a "nicked oligonucleotide"), with the presence of such a discontinuity predicted to direct RNase H cleavage to form double-stranded cleavage products having a precise structure. Specifically, the presence of a discontinuity within the first strand of the bifunctional DsiRNA agent depicted in FIG. 1 effectively primes the bifunctional DsiRNA agent for cleavage by RNase H, as the RNase H enzyme need only cleave the unmodified ribonucleotide-containing strand ("the RNase H-substrate domain") that does not possess such discontinuity in order to liberate two independent RNase H cleavage products (in FIG. 1, the second strand is the continuous, "non-nicked" strand, and is the only strand that RNase H need cleave in order to liberate two independent Dicer substrate molecules; it is noted in FIG. 1 that the second strand of the second region's most 5' ribonucleotides are modified ribonucleotides, thereby directing RNase H cleavage to occur 3' of these modified ribonucleotides of the second strand, thereby liberating a cleavage product of the second and third regions that possesses a 3' overhang of two modified ribonucleotides on the second strand (refer to right-hand RNase H cleavage product of FIG. 1)).

[0131] In an alternate embodiment of the bifunctional DsiRNA agent shown in FIG. 1, labelled sections 2 and 6 of FIG. 1 might effectively be swapped, resulting in an agent possessing a discontinuity (nick) to the immediate 5' of the 5' terminal deoxyribonucleotide of the second strand (with labelled section 6 comprising deoxyribonucleotides and constituting an "RNase H-activating domain") while labelled section 2 comprises at least four unmodified ribonucleotides that base pair with each of the four deoxyribonucleotides now constituting labelled section 6 (with such unmodified nucleotides of labelled section 2 now constituting an "RNase H-substrate domain"). As the skilled artisan will recognize, these RNase H-activating domains and RNase H-substrate domains can optionally be extended or modified, so long as this region retains its character as an RNase H-cleavable domain, with RNase H cleavage resulting in liberation of two independent Dicer substrate agents.

[0132] FIG. 2 depicts a double-stranded oligonucleotide synthesized to possess the following structure: (1) a first region having a RISC-activating domain (preferably dsRNA) of about 23 to 33 duplexed nucleotides in length (the agent shown in FIG. 2 specifically possesses a 23 nucleotide length of duplexed dsRNAs and a 2 nucleotide length of duplexed deoxyribonucleotides at the 3' end of the first strand/5' end of the second strand of this first region of the bifunctional DsiRNA agent); (2) a second region that includes an RNA:DNA hybrid domain of at least four duplexed nucleotides in length, with such RNA:DNA hybrid domain constituting an RNase H-cleavable site (the agent shown in FIG. 2 specifically possesses an RNA:DNA hybrid region of 4 duplexed nucleotides in length, with the ribonucleotides of this 4 base pair RNA:DNA span being unmodified ribonucleotides (the deoxyribonucleotides of this region may be either modified or unmodified deoxyribonucleotides)--presence of DNA:DNA base pairs in those parts of the first and third regions of the bifunctional DsiRNA agent immediately flanking this RNA:DNA span are predicted to direct RNase H cleavage away from such DNA:DNA domains, instead directing such RNase H cleavage to occur at a location(s) within the (unmodified RNA):DNA duplexed nucleotides of this second region; and (3) a third region having a RISC-activating domain (preferably dsRNA) of about 23 to 33 duplexed nucleotides in length (the agent shown in FIG. 2 specifically possesses a 23 nucleotide length of duplexed dsRNAs and a two base pair length DNA:DNA duplex at the 5' terminus of the first strand/3' terminus of the second strand of this third region of the bifunctional DsiRNA agent).

[0133] As with the agent depicted in FIG. 1, the first region of an RNase H-cleavable agent such as the one depicted in FIG. 2 can optionally comprise an overhang (optionally a 3' overhang of 1-4 nucleotides in length). In certain embodiments, the nucleotides of such an overhang are modified ribonucleotides, or they may comprise deoxyribonucleotides. In addition, the duplexed nucleotides of the first region can also comprise modified nucleotides, e.g., modified ribonucleotides, e.g., at alternating locations within the span of the second strand that is complementary to a target RNA and that is modeled to lie 3' of the Dicer cleavage site of the DsiRNA agent that is predicted to be formed from the first and second regions via RNase H cleavage of the bifunctional (precursor) RNase H-cleavable DsiRNA-containing starting agent (bifunctional DsiRNA agent). Indeed, the RNase H-processed products of the bifunctional agent shown in FIG. 2 are, in turn, processed by Dicer to yield two independent, active siRNA agents.

[0134] In view of the third region of the above-described RNase H-cleavable "bifunctional DsiRNA" also comprising a RISC-activating domain that is liberated, optionally in concert with a portion of the second region, to form a DsiRNA agent via RNase H cleavage, the nucleotides of this third region may also comprise modified nucleotides, e.g., modified ribonucleotides. In certain embodiments, such modified nucleotides are positioned at alternating locations within the span of the first strand that is complementary to a target RNA and that is modeled to lie 3' on the first strand from the Dicer cleavage site of the DsiRNA agent that is predicted to be formed from the second and third regions via RNase H cleavage of the bifunctional (precursor) RNase H-cleavable DsiRNA-containing starting agent. It is noted that the first strand of the bifunctional DsiRNA agent depicted in FIG. 2 also possesses a discontinuity (nick), with the presence of such a discontinuity predicted to direct RNase H cleavage to form double-stranded cleavage products having a precise structure. Specifically, the presence of a discontinuity within the first strand of the bifunctional DsiRNA agent depicted in FIG. 2 effectively primes the bifunctional DsiRNA agent for cleavage by RNase H, as the RNase H enzyme need only cleave the unmodified ribonucleotide-containing strand ("the RNase H-substrate domain") that does not possess such discontinuity in order to liberate two independent RNase H cleavage products (in FIG. 2, the second strand is the continuous, "non-nicked" strand, and is the only strand that RNase H need cleave in order to liberate two independent Dicer substrate molecules; it is noted in FIG. 2 that the most 5' nucleotides of the second strand of the first region are deoxyribonucleotides (as are the cognate most 3' nucleotides of the first strand of the first region), thereby directing RNase H cleavage to occur 5' of these deoxyribonucleotides of the second strand, thereby liberating a cleavage product of the first region that possesses two deoxyribonucleotides at the 3' terminus of the first strand that base pair with two cognate deoxyribonucleotides of the second strand. Even if RNase H cleavage occurs upstream of these deoxyribonucleotides of the second strand, any single stranded RNAs should be degraded ("processive"), yielding a blunt ended DsiRNA agent at the 3' terminus of the first strand/5' terminus of the second strand. Meanwhile, liberation of a DsiRNA agent from the second and third regions of the bifunctional agent shown in FIG. 2 is modeled to involve RNase H cleavage, followed by degradation of ribonucleotides of the second region, resulting in an RNase H-liberated agent that possesses a 5' overhang of deoxyribonucleotides and is subsequently processed by Dicer. As noted above in reference to the structure of the bifunctional DsiRNA agent of FIG. 2, the DsiRNA agent formed from regions 2 and 3 that is liberated via RNase H cleavage optionally contains modified nucleotides, e.g., modified ribonucleotides, e.g., positioned at the 3' overhang nucleotides of the first strand and/or present at alternating residues within the span of nucleotides of the first strand that are predicted to be 3' of the eventual Dicer cleavage site, in an agent having a structure such as that shown in FIG. 2.

[0135] In alternate embodiments of the bifunctional DsiRNA agent shown in FIG. 2, the RNA:DNA region and location of discontinuity of the bifunctional DsiRNA agent can be exchanged between the two strands. In a vertical flipping (inversion) of RNA/DNA identity within such an RNA:DNA region possessing discontinuity, a resultant structure has RNA residues on the first strand within the second region, with such first strand being continuous across the expanse of the bifunctional DsiRNA agent, while the second strand of the second region would comprise cognate DNA residues, with the second strand (rather than the first strand) now possessing a discontinuity that is located at the immediate edge of the RNA:DNA span of duplexed nucleotides (with such nick located at the 3' end of the second strand of the second region).

[0136] As will be clear to the skilled artisan, the position of the strand discontinuity (nick) shown or described in many of the agents recited herein may be altered so long as activity of such agents is retained. It is noted for the agent shown in FIG. 1 that the positioning of a nick in the first strand deoxyribonucleotide span as indicated directs RNase H cleavage such that a two ribonucleotide 3' overhang is created in the released DsiRNA agent comprising labelled domains 3, 4 and 7 of the FIG. 1 agent. However, in the agent shown in FIG. 1, a nick in the first strand deoxyribonucleotide span could also be introduced at any of the following positions, even were the activity of the released DsiRNAs to be less effective than the optimized nicked version shown (nick is indicated in the following sequences by a vertical line, "|"): 5'- . . . AA|ttcaccggGGA . . . -3'; 5'- . . . AAt|tcaccggGGA . . . -3'; 5'- . . . AAtt|caccggGGA . . . -3'; 5'- . . . AAttc|accggGGA . . . -3'; 5'- . . . AAttca|ccggGGA . . . -3'; 5'- . . . AAttcac|cggGGA . . . -3'; 5'- . . . AAttcacc|ggGGA . . . -3'; 5'- . . . AAttcaccg|gGGA . . . -3'; 5'- . . . AAttcaccgg|GGA . . . -3'. Similarly, for an agent of FIG. 1 that is altered to swap the location of DNA and RNA residues within the RNA:DNA duplex domain, a nick in the resultant second strand's deoxyribonucleotide span can be introduced at any of the following positions (nick is indicated in the following sequences by a vertical line, "|"): 5'- . . . UCC|ccggugaaUUU . . . -3'; 5'- . . . UCCc|cggugaaUUU . . . -3'; 5'- . . . UCCcc|ggugaaUUU . . . -3'; 5'- . . . UCCccg|gugaaUUU . . . -3'; 5'- . . . UCCccgg|ugaaUUU . . . -3'; 5'- . . . UCCccggu|gaaUUU . . . -3'; 5'- . . . UCCccggug|aaUUU . . . -3'; ; 5'- . . . UCCccgguga|aUUU . . . -3'; ; 5'- . . . UCCccggugaa|UUU . . . -3'. It is also possible to introduce two or more nicks within the deoxyribonucleotide stretches of any such domain, e.g., more precise definition of DsiRNA structures generated via RNase H cleavage of the agent shown in FIG. 1 might be obtained via inclusion of nicks at the following locations of the first strand: 5'- . . . AAtt|caccgg|GGA . . . -3'.

[0137] As for bifunctional agents of the invention that possess structures as shown in or akin to those of FIG. 1, bifunctional agents such as those shown in FIG. 2 can also harbor a discontinuity at any of a number of positions within the strand that possesses the deoxyribonucleotides of the RNA:DNA duplexed nucleotides which attract RNase H cleavage. Specifically, within the agent shown in FIG. 2, such a discontinuity in the first strand can be introduced at any of the following positions (nick is indicated in the following sequences by a vertical line, "|"): 5'- . . . AAA|ttcaccggUCG . . . -3'; 5'- . . . AAAt|tcaccggUCG . . . -3'; 5'- . . . AAAtt|caccggUCG . . . -3'; 5'- . . . AAAttc|accggUCG . . . -3'; 5'- . . . AAAttca|ccggUCG . . . -3'; 5'- . . . AAAttcac|cggUCG . . . -3'; 5'- . . . AAAttcacc|ggUCG . . . -3'; 5'- . . . AAAttcaccg|gUCG . . . -3'; 5'- . . . AAAttcaccgg|UCG . . . -3'. In an agent such as that shown in FIG. 2, yet in which RNA and DNA positions of the RNA:DNA duplex domain have been swapped, a discontinuity can correspondingly be introduced into the second strand at any of the following positions (nick is indicated in the following sequences by a vertical line, "|"): 5'- . . . CGA|ccggugaaUUU . . . -3'; 5'- . . . CGAc|cggugaaUUU . . . -3'; 5'- . . . CGAcc|ggugaaUUU . . . -3'; 5'- . . . CGAccg|gugaaUUU . . . -3'; 5'- . . . CGAccgg|ugaaUUU . . . -3'; 5'- . . . CGAccggu|gaaUUU . . . -3'; 5'- . . . CGAccggug|aaUUU . . . -3'; 5'- . . . CGAccgguga|aUUU . . . -3'; 5'- . . . CGAccggugaa|UUU . . . -3'

[0138] Bifunctional agents of the invention can also be generated without nicks, with RNase H cleavage releasing DsiRNA agents that retain activity.

[0139] RNase H-cleavable RNA:DNA duplex domains can also be used to tether releasable functional groups/payloads to double-stranded nucleotides harboring a DsiRNA agent which is released upon RNase H cleavage. Specifically, FIG. 3 shows a composition having: (1) a first region comprising dsRNA; (2) a second region comprising an RNA:DNA duplex domain, within which at least 4 ribonucleotides of such region are unmodified ribonucleotides and (3) a functional group/payload that is attached to the RNA-containing strand of the RNA:DNA duplex domain of the second region. Such a functional group/payload may be attached via an art-recognized covalent or non-covalent linkage. The DNA-containing strand of the agent shown in FIG. 3 may optionally contain a discontinuity, e.g., at 5'- . . . GAAAtt|caccgg-3'.

[0140] Many of the nucleotide modifications (e.g., 2'-O-methyl groups, LNA, etc.) described herein can impact upon properties such as biodistribution, formulation, adsorption, metabolism, pharmacodynamic, cellular uptake, etc. of the double stranded nucleic acid agents of the invention. In one aspect, the invention features double stranded nucleic acid molecules having improved qualities (e.g., bioavailability, cellular uptake, etc.) imparted via tethering of a functional group to the structure of a (DsiRNA-containing) double stranded nucleic acid (see, e.g., FIG. 3). Altered bioavailability or other properties attributable to presence of such a functional group within a double stranded nucleic acid of the invention can be assessed under conditions suitable for isolating double stranded nucleic acid molecules having improved bioavailability or other such properties. Such functional groups can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; polyamines, such as spermine or spermidine; and others.

[0141] Functional groups of the invention can include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmaco-dynamic properties of nucleic acid agents, and groups that enhance the pharmacokinetic properties of nucleic acid agents. Typical functional groups include cholesterols, lipids, phospho lipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, enhance RISC residency and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative functional groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860. Functional group moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Nucleic acid agents of the invention may also be attached to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999). Representative United States patents that teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0142] Non-limiting further examples of functional groups contemplated by the invention include conjugates and ligands described in Vargeese et al., U.S. Application No. 2004/0110296. In one embodiment, a functional group 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 functional group attached to the double stranded nucleic acid molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific functional groups contemplated by the instant invention that can be attached to double stranded nucleic acids within the agents of the invention are described, e.g., in Vargeese et al., U.S. Application No. 2003/0130186. The type and number of functional groups/conjugates used in a double stranded nucleic acid of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of double stranded nucleic acid constructs while at the same time maintaining the ability of the double stranded nucleic acid to mediate RNAi activity. As such, one skilled in the art can screen double stranded nucleic acid constructs that are modified with various functional groups/conjugates to determine whether the double stranded nucleic acid-functional group agent possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.

[0143] The second region of any of the above-recited agents can be extended in length, e.g., such that the RNA:DNA duplex domain has a length of 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. Optionally, the length of such RNA:DNA duplex domain is of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 nucleotides in length.

[0144] In one embodiment, the RNA:DNA duplex domain of the double stranded nucleic acid agent of the instant invention can comprise an antisense agent. In such embodiments, it is particularly advantageous that the RNA:DNA duplex domain length be of at least about 12 nucleotides in length. Where such a region includes an antisense agent that is releasable upon RNase H cleavage of the RNase H-cleavable starting agent of the instant invention, modifications of the antisense deoxyribonucleotide domain can be performed, including any such art-recognized modification of antisense agents that has been described. Exemplary modifications of such an antisense agent harbored within the RNA:DNA duplex domain of a double stranded nucleic acid of the invention include phosphorothioate modification of such a deoxynucleotide region, with, e.g., inclusion of other (additional) modifications also allowed. For example, a 12mer antisense agent may be included within the RNA:DNA duplex domain of a double stranded nucleic acid of the invention, with such DNAs of said RNA:DNA duplex domain comprising phosphorothioate modifications (such modifications are notably still capable of activating RNase H cleavage), with such deoxyribonucleotide sequence optionally possessing LNA moieties at each end of the deoxyribonucleotide domain, e.g., such that an 8mer of unmodified DNA and/or only phosphorothioate-modified deoxyribonucleotide residues are located at the center of such 12mer domain that activates RNase H. Exemplary modification patterns that can be used within antisense agents contained within double stranded nucleic acid agents of the instant invention can be found, e.g., within U.S. Pat. No. 7,432,250. It is noted that in the context of a bifunctional RNase H-cleavable double stranded nucleic acid of the instant invention, such antisense agent-containing double stranded nucleic acid of the invention can also be referred to as "trifunctional" (specifically, RNase H cleavage not only releases two, optionally independent, DsiRNA agents, but also releases an antisense agent from the RNase H-cleavable RNA:DNA region).

[0145] Following RNase H cleavage of the bifunctional and functional group-tethered DsiRNA agents of the instant invention, Dicer enzyme is predicted to bind to liberated DsiRNA agents, resulting in cleavage of such DsiRNAs at a position 19-23 nucleotides removed from a Dicer PAZ domain-associated 3' overhang sequence of the antisense strand of the DsiRNA agent. This Dicer cleavage event results in excision of those duplexed nucleic acids previously located at the 3' end of the passenger (sense) strand and 5' end of the guide (antisense) strand. (Cleavage of the DsiRNA typically yields a 19mer duplex with 2-base overhangs at each end.) As presently modeled in FIGS. 1-3, this Dicer cleavage event generates a 21-23 nucleotide guide (antisense) strand capable of directing sequence-specific inhibition of target mRNA as a RISC component.

[0146] The first and second oligonucleotide strands of the bifunctional and functional group-tethered DsiRNA agents of the instant invention are not required to be completely complementary. In fact, in one embodiment, the 3'-terminus of the sense strand of a constituent DsiRNA agent contains one or more mismatches. In one aspect, about two mismatches are incorporated at the 3' terminus of the sense (passenger) strand. In another embodiment, the constituent DsiRNA(s) of the invention are a double stranded RNA molecule containing two RNA oligonucleotides each of which is an identical number of nucleotides in the range of 27-35 nucleotides in length and, when annealed to each other, have blunt ends and a two nucleotide mismatch on the 3'-terminus of the sense strand (the 5'-terminus of the antisense strand). The use of mismatches or decreased thermodynamic stability (specifically at the 3'-sense/5'-antisense position) has been proposed to facilitate or favor entry of the antisense strand into RISC (Schwarz et al., 2003; Khvorova et al., 2003), presumably by affecting some rate-limiting unwinding steps that occur with entry of the siRNA into RISC. Thus, terminal base composition has been included in design algorithms for selecting active 21mer siRNA duplexes (Ui-Tei et al., 2004; Reynolds et al., 2004). With Dicer cleavage of the dsRNA region of this embodiment, the small end-terminal sequence which contains the mismatches will either be left unpaired with the antisense strand (become part of a 3'-overhang) or be cleaved entirely off the final 21-mer siRNA. These "mismatches", therefore, do not persist as mismatches in the final RNA component of RISC. The finding that base mismatches or destabilization of segments at the 3'-end of the sense strand of Dicer substrate improved the potency of synthetic duplexes in RNAi, presumably by facilitating processing by Dicer, was a surprising finding of past works describing the design and use of 25-30mer dsRNAs (also termed "DsiRNAs" herein; Rossi et al., U.S. Patent Application Nos. 2005/0277610, 2005/0244858 and 2007/0265220). DsiRNAs having base-paired deoxyribonucleotides at passenger (sense) strand positions modeled to be 3' of the Dicer cleavage site have also been identified as at least equally effective as RNA-RNA duplex-extended DsiRNA agents (U.S. Patent Application No. 61/138,946, filed Dec. 18, 2008). Thus, dsDNA-extended DsiRNA agents such as those described in U.S. Patent Application No. 61/138,946 may also be incorporated as constituent DsiRNA agents within the RNase H-cleavable constructs of the instant invention.

[0147] As shown in FIG. 4, bifunctional DsiRNA agents can be synthesized that do not require a RNA:DNA RNase H cleavable joining sequence to function as a bifunctional agent. Rather, a double stranded DNA:DNA extended region of the DsiRNAs comprising such an agent provides the joining sequence for the bifunctional agent. The second region of such an agent can be extended in length, e.g., such that the DNA:DNA duplex domain has a length of 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 nucleotides in length. Optionally, the length of such DNA:DNA duplex domain is of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 nucleotides in length. Such a dsDNA extended joining sequence may also be modified as described above, or may provide an antisense agent within the dsDNA extended region, imparting "trifunctionality" to such a dsDNA extended/joined agent.

[0148] In certain embodiments, a bifunctional inhibitory agent of the invention is cleaved by Dicer following administration to a cell, tissue and/or subject. In such aspects, the bifunctional agents of the instant invention possess enhanced efficacy and/or potency as compared to tandem siRNA agents that have previously been described. Indeed, such bifunctional agents of the instant invention can be capable of inhibiting expression of both targeted genes (or, where the bifunctional agent targets two or more sites within the same gene, of the single targeted gene) by at least 20% at a concentration of 100 picomolar in the environment of a cell. In certain embodiments, levels of both targeted genes are reduced by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% or more at a selected sub-nanomolar concentration in the environment of a cell (e.g., 500 pM or less, 200 pM or less, 100 pM or less, 50 pM or less, 25 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, 2 pM or less, or 1 pM or less). Such surprising in vitro efficacies and/or potencies can also correspond to enhanced in vivo efficacies and/or potencies for such bifunctional agents of the instant invention, with respect to an appropriate control molecule (e.g., tandem siRNAs) and/or pair of control molecules (e.g., siRNAs that are not attached to one another).

Exemplary Structures of Constituent DsiRNAs of RNase H-Cleavable DsiRNA Agent Compositions and dsDNA Extended Bifunctional Agents

[0149] In one aspect, the present invention provides compositions for RNA interference (RNAi) that comprise DsiRNA agent(s) that are liberated from functional group(s) or additional DsiRNA agents via RNase H cleavage of a RNA:DNA duplex-containing RNase H cleavable region. (Alternatively, DsiRNA moieties are joined by a double stranded DNA joining sequence in a bifunctional dsDNA extended agent.) Certain compositions of the invention comprise a double stranded nucleic acid which is a precursor molecule, i.e., the double stranded nucleic acid of the present invention is initially processed in vitro or in vivo by RNase H to yield one or more DsiRNA agents. Such liberated DsiRNA agents are, in turn, processed in vivo or in cells (or in an in vitro Dicer cleavage assay) to produce an active small interfering nucleic acid (siRNA). The double stranded nucleic acid is processed by Dicer to an active siRNA which is incorporated into RISC.

[0150] In certain embodiments, the constituent DsiRNA component agents of the bifunctional or functional group-tethered DsiRNA agents of the invention can have any of the following exemplary structures:

[0151] In one embodiment, the constituent DsiRNA agent(s) comprises:

TABLE-US-00002 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0152] In another such embodiment, the constituent DsiRNA agent(s) comprises:

TABLE-US-00003 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0153] In another such embodiment, the constituent DsiRNA agent(s) comprises:

TABLE-US-00004 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0154] In another embodiment, the constituent DsiRNA agent(s) comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the constituent DsiRNA, when numbering from the 3'-terminal residue, are mismatched with corresponding residues of the 5'-terminal region on the second strand when first and second strands are annealed to one another). An exemplary 27mer constituent DsiRNA agent with two terminal mismatched residues is shown:

TABLE-US-00005 5'-pXXXXXXXXXXXXXXXXXXXXXXXXX.sup.M.sup.M-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXX.sub.M.sub.Mp-5'

wherein "X"=RNA, "p"=a phosphate group, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers--alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. The top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand.

[0155] In one embodiment, the constituent DsiRNA agent has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 1-4 base 3'-overhang (e.g., a one base 3'-overhang, a two base 3'-overhang, a three base 3'-overhang or a four base 3'-overhang). In another embodiment, this DsiRNA agent has an asymmetric structure further containing 2 deoxynucleotides at the 3' end of the sense strand.

[0156] In another embodiment, the constituent DsiRNA agent has an asymmetric structure, with the antisense strand having a 25-base pair length, and the sense strand having a 27-base pair length with a 1-4 base 3'-overhang (e.g., a one base 3'-overhang, a two base 3'-overhang, a three base 3'-overhang or a four base 3'-overhang). In another embodiment, this DsiRNA agent has an asymmetric structure further containing 2 deoxynucleotides at the 3' end of the antisense strand.

[0157] In additional embodiments, one or more constituent DsiRNA(s) can comprise dsDNA-extended ("DNA handle") structures, such as:

TABLE-US-00006 5'-XXXXXXXXXXXXXXXXXXXXXXXD.sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXD.sub.NXX-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, and "N"=1 to 50 or more, but is optionally 1-8. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

[0158] In a related embodiment, the constituent DsiRNA comprises:

TABLE-US-00007 5'-XXXXXXXXXXXXXXXXXXXXXXXD.sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXD.sub.NDD-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, and "N"=1 to 50 or more, but is optionally 1-8. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

[0159] In another such embodiment, the constituent DsiRNA comprises:

TABLE-US-00008 5'-XXXXXXXXXXXXXXXXXXXXXXXD.sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXD.sub.NZZ-5'

wherein "X"=RNA, "X"=2'-O-methyl RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=1 to 50 or more, but is optionally 1-8. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0160] In another such embodiment, the constituent DsiRNA comprises:

TABLE-US-00009 5'-XXXXXXXXXXXXXXXXXXXXXXXD.sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXD.sub.NZZ-5'

wherein "X"=RNA, "X"=2'-O-methyl RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=1 to 50 or more, but is optionally 1-8. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0161] In another embodiment, the constituent DsiRNA comprises:

TABLE-US-00010 5'-XXXXXXXXXXXXXXXXXXXXXXX[X1/D1].sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXX[X2/D2].sub.NZZ-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=1 to 50 or more, but is optionally 1-8, where at least one D1.sub.N is present in the top strand and is base paired with a corresponding D2.sub.N in the bottom strand. Optionally, D1.sub.N and D1.sub.N+1 are base paired with corresponding D2.sub.N and D2.sub.N+1; D1.sub.N; D1.sub.N+1 and D1.sub.N+2 are base paired with corresponding D2.sub.N, D1.sub.N+1 and D1.sub.N+2, etc. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0162] In any of the above-depicted structures, the 5' end of either the sense strand or antisense strand optionally comprises a phosphate group.

[0163] In another embodiment, the constituent DNA:DNA-extended DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the constituent DsiRNA, when numbering from the 3'-terminal residue, are mismatched with corresponding residues of the 5'-terminal region on the second strand when first and second strands are annealed to one another). An exemplary constituent DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown:

TABLE-US-00011 5'-XXXXXXXXXXXXXXXXXXXXXXXXXD.sub.N.sup.M.sup.M-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXD.sub.NM.sub.M-5'

wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed, "D"=DNA and "N"=1 to 50 or more, but is optionally 1-8. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers--alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. In one embodiment, the top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric/overhang agents can also be incorporated into such "blunt/frayed" agents.

[0164] In one embodiment, a length-extended constituent DsiRNA agent is provided that comprises deoxyribonucleotides positioned at sites modeled to function via specific direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the double stranded nucleic acid structure. An exemplary structure for such a molecule is shown:

TABLE-US-00012 5'-XXXXXXXXXXXXXXXXXXXDDXX-3' 3'-YXXXXXXXXXXXXXXXXXDDXXXX-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "D"=DNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. The above structure is modeled to force Dicer to cleave a minimum of a 21mer duplex as its primary post-processing form. In embodiments where the bottom strand of the above structure is the antisense strand, the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off-target effects (as prior studies have shown a 2'-O-methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007/0223427).

[0165] In certain embodiments, the "D" residues of any of the above structures include at least one PS-DNA or PS-RNA. Optionally, the "D" residues of any of the above structures include at least one modified nucleotide that inhibits Dicer cleavage.

[0166] In some embodiments, the constituent DsiRNA agent of the instant invention further comprises a linking moiety or domain that joins the sense and antisense strands of a constituent DNA:DNA-extended DsiRNA agent. Optionally, such a linking moiety domain joins the 3' end of the sense strand and the 5' end of the antisense strand. The linking moiety may be a chemical (non-nucleotide) linker, such as an oligomethylenediol linker, oligoethylene glycol linker, or other art-recognized linker moiety. Alternatively, the linker can be a nucleotide linker, optionally including an extended loop and/or tetraloop.

[0167] In one embodiment, the constituent DsiRNA agent has an asymmetric structure, with the sense strand having a 27-base pair length, the antisense strand having a 29-base pair length with a 1-4 base 3'-overhang (e.g., a one base 3'-overhang, a two base 3'-overhang, a three base 3'-overhang or a four base 3'-overhang), and with deoxyribonucleotides located at positions 24 and 25 of the sense strand (numbering from position 1 at the 5' of the sense strand) and each base paired with a cognate deoxyribonucleotide of the antisense strand. In another embodiment, this constituent DsiRNA agent has an asymmetric structure further containing 2 deoxyribonucleotides at the 3' end of the sense strand.

Modification of Constituent DsiRNA(s) of Bifunctional or Functional Group-Tethered DsiRNA Agents

[0168] One major factor that inhibits the effect of double stranded RNAs ("dsRNAs") is the degradation of dsRNAs (e.g., siRNAs and DsiRNAs) by nucleases. A 3'-exonuclease is the primary nuclease activity present in serum and modification of the 3'-ends of antisense DNA oligonucleotides is crucial to prevent degradation (Eder et al., 1991). An RNase-T family nuclease has been identified called ERI-1 which has 3' to 5' exonuclease activity that is involved in regulation and degradation of siRNAs (Kennedy et al., 2004; Hong et al., 2005). This gene is also known as Thex1 (NM.sub.--02067) in mice or THEX1 (NM.sub.--153332) in humans and is involved in degradation of histone mRNA; it also mediates degradation of 3'-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al., 2006). It is therefore reasonable to expect that 3'-end-stabilization of dsRNAs, including the DsiRNAs of the instant invention, will improve stability.

[0169] XRN1 (NM.sub.--019001) is a 5' to 3' exonuclease that resides in P-bodies and has been implicated in degradation of mRNA targeted by miRNA (Rehwinkel et al., 2005) and may also be responsible for completing degradation initiated by internal cleavage as directed by a siRNA. XRN2 (NM.sub.--012255) is a distinct 5' to 3' exonuclease that is involved in nuclear RNA processing. Although not currently implicated in degradation or processing of siRNAs and miRNAs, these both are known nucleases that can degrade RNAs and may also be important to consider.

[0170] RNase A is a major endonuclease activity in mammals that degrades RNAs. It is specific for ssRNA and cleaves at the 3'-end of pyrimidine bases. SiRNA degradation products consistent with RNase A cleavage can be detected by mass spectrometry after incubation in serum (Turner et al., 2007). The 3'-overhangs enhance the susceptibility of siRNAs to RNase degradation. Depletion of RNase A from serum reduces degradation of siRNAs; this degradation does show some sequence preference and is worse for sequences having poly A/U sequence on the ends (Haupenthal et al., 2006). This suggests the possibility that lower stability regions of the duplex may "breathe" and offer transient single-stranded species available for degradation by RNase A. RNase A inhibitors can be added to serum and improve siRNA longevity and potency (Haupenthal et al., 2007).

[0171] In 21mers, phosphorothioate or boranophosphate modifications directly stabilize the internucleoside phosphate linkage. Boranophosphate modified RNAs are highly nuclease resistant, potent as silencing agents, and are relatively non-toxic. Boranophosphate modified RNAs cannot be manufactured using standard chemical synthesis methods and instead are made by in vitro transcription (IVT) (Hall et al., 2004 and Hall et al., 2006). Phosphorothioate (PS) modifications can be readily placed in an RNA duplex at any desired position and can be made using standard chemical synthesis methods, though the ability to use such modifications within an RNA duplex that retains RNA silencing activity can be limited. Because PS moieties are likely to require greater spacing when included within an RNA duplex-containing agent in order to retain RNA inhibitory activity, dsDNA extension of constituent DsiRNAs such as those described herein can provide a means of including more PS modifications (either PS-DNA or PS-RNA) within a single constituent DsiRNA agent than would otherwise be available were no such extension used. It is noted, however, that the PS modification shows dose-dependent toxicity, so most investigators have recommended limited incorporation in siRNAs, historically favoring the 3'-ends where protection from nucleases is most important (Harborth et al., 2003; Chiu and Rana, 2003; Braasch et al., 2003; Amarzguioui et al., 2003). More extensive PS modification can be compatible with potent RNAi activity; however, use of sugar modifications (such as 2'-O-methyl RNA) may be superior (Choung et al., 2006).

[0172] A variety of substitutions can be placed at the 2'-position of the ribose which generally increases duplex stability (T.sub.m) and can greatly improve nuclease resistance. 2'-O-methyl RNA is a naturally occurring modification found in mammalian ribosomal RNAs and transfer RNAs. 2'-O-methyl modification in siRNAs is known, but the precise position of modified bases within the duplex is important to retain potency and complete substitution of 2'-O-methyl RNA for RNA will inactivate the siRNA. For example, a pattern that employs alternating 2'-O-methyl bases can have potency equivalent to unmodified RNA and is quite stable in serum (Choung et al., 2006; Czauderna et al., 2003).

[0173] The 2'-fluoro (2'-F) modification is also compatible with dsRNA (e.g., siRNA and DsiRNA) function; it is most commonly placed at pyrimidine sites (due to reagent cost and availability) and can be combined with 2'-O-methyl modification at purine positions; 2'-F purines are available and can also be used. Heavily modified duplexes of this kind can be potent triggers of RNAi in vitro (Allerson et al., 2005; Prakash et al., 2005; Kraynack and Baker, 2006) and can improve performance and extend duration of action when used in vivo (Morrissey et al., 2005a; Morrissey et al., 2005b). A highly potent, nuclease stable, blunt 19mer duplex containing alternative 2'-F and 2'-O-Me bases is taught by Allerson. In this design, alternating 2'-O-Me residues are positioned in an identical pattern to that employed by Czauderna, however the remaining RNA residues are converted to 2'-F modified bases. A highly potent, nuclease resistant siRNA employed by Morrissey employed a highly potent, nuclease resistant siRNA in vivo. In addition to 2'-O-Me RNA and 2'-F RNA, this duplex includes DNA, RNA, inverted abasic residues, and a 3'-terminal PS internucleoside linkage. While extensive modification has certain benefits, more limited modification of the duplex can also improve in vivo performance and is both simpler and less costly to manufacture. Soutschek et al. (2004) employed a duplex in vivo and was mostly RNA with two 2'-O-Me RNA bases and limited 3'-terminal PS internucleoside linkages.

[0174] Locked nucleic acids (LNAs) are a different class of 2'-modification that can be used to stabilize dsRNA (e.g., siRNA and DsiRNA). Patterns of LNA incorporation that retain potency are more restricted than 2'-O-methyl or 2'-F bases, so limited modification is preferred (Braasch et al., 2003; Grunweller et al., 2003; Elmen et al., 2005). Even with limited incorporation, the use of LNA modifications can improve dsRNA performance in vivo and may also alter or improve off target effect profiles (Mook et al., 2007).

[0175] Synthetic nucleic acids introduced into cells or live animals can be recognized as "foreign" and trigger an immune response. Immune stimulation constitutes a major class of off-target effects which can dramatically change experimental results and even lead to cell death. The innate immune system includes a collection of receptor molecules that specifically interact with DNA and RNA that mediate these responses, some of which are located in the cytoplasm and some of which reside in endosomes (Marques and Williams, 2005; Schlee et al., 2006). Delivery of siRNAs by cationic lipids or liposomes exposes the siRNA to both cytoplasmic and endosomal compartments, maximizing the risk for triggering a type 1 interferon (IFN) response both in vitro and in vivo (Morrissey et al., 2005b; Sioud and Sorensen, 2003; Sioud, 2005; Ma et al., 2005). RNAs transcribed within the cell are less immunogenic (Robbins et al., 2006) and synthetic RNAs that are immunogenic when delivered using lipid-based methods can evade immune stimulation when introduced unto cells by mechanical means, even in vivo (Heidel et al., 2004). However, lipid based delivery methods are convenient, effective, and widely used. Some general strategy to prevent immune responses is needed, especially for in vivo application where all cell types are present and the risk of generating an immune response is highest. Use of chemically modified RNAs may solve most or even all of these problems.

[0176] Although certain sequence motifs are clearly more immunogenic than others, it appears that the receptors of the innate immune system in general distinguish the presence or absence of certain base modifications which are more commonly found in mammalian RNAs than in prokaryotic RNAs. For example, pseudouridine, N6-methyl-A, and 2'-O-methyl modified bases are recognized as "self" and inclusion of these residues in a synthetic RNA can help evade immune detection (Kariko et al., 2005). Extensive 2'-modification of a sequence that is strongly immunostimulatory as unmodified RNA can block an immune response when administered to mice intravenously (Morrissey et al., 2005b). However, extensive modification is not needed to escape immune detection and substitution of as few as two 2'-O-methyl bases in a single strand of a siRNA duplex can be sufficient to block a type 1 IFN response both in vitro and in vivo; modified U and G bases are most effective (Judge et al., 2006). As an added benefit, selective incorporation of 2'-O-methyl bases can reduce the magnitude of off-target effects (Jackson et al., 2006). Use of 2'-O-methyl bases should therefore be considered for all dsRNAs intended for in vivo applications as a means of blocking immune responses and has the added benefit of improving nuclease stability and reducing the likelihood of off-target effects.

[0177] Although cell death can result from immune stimulation, assessing cell viability is not an adequate method to monitor induction of IFN responses. IFN responses can be present without cell death, and cell death can result from target knockdown in the absence of IFN triggering (for example, if the targeted gene is essential for cell viability). Relevant cytokines can be directly measured in culture medium and a variety of commercial kits exist which make performing such assays routine. While a large number of different immune effector molecules can be measured, testing levels of IFN-.alpha., TNF-.alpha., and IL-6 at 4 and 24 hours post transfection is usually sufficient for screening purposes. It is important to include a "transfection reagent only control" as cationic lipids can trigger immune responses in certain cells in the absence of any nucleic acid cargo. Including controls for IFN pathway induction should be considered for cell culture work. It is essential to test for immune stimulation whenever administering nucleic acids in vivo, where the risk of triggering IFN responses is highest.

[0178] Modifications can be included in the bifunctional or functional group-tethered DsiRNA agents of the present invention so long as the modification does not prevent the bifunctional or functional group-tethered DsiRNA agent from serving as a substrate for RNase H, and so long as such modification also does not prevent constituent DsiRNA agent(s) that are liberated post-RNase H cleavage from serving as a substrate for Dicer. As recently described in U.S. Patent Application 61/138,946, base paired deoxyribonucleotides can be attached to DsiRNA molecules, resulting in enhanced RNAi efficacy and duration, provided that such extension is performed in a region of the extended molecule that does not interfere with Dicer processing (e.g., 3' of the Dicer cleavage site of the sense strand/5' of the Dicer cleavage site of the antisense strand). In one embodiment, one or more modifications are made that enhance Dicer processing of the constituent DsiRNA agent(s) of a bifunctional or functional group-tethered DsiRNA agent of the invention. In a second embodiment, one or more modifications are made that result in more effective RNAi generation. In a third embodiment, one or more modifications are made that support a greater RNAi effect. In a fourth embodiment, one or more modifications are made that result in greater potency per each constituent DsiRNA agent molecule to be delivered to the cell. Modifications can be incorporated in the 3'-terminal region, the 5'-terminal region, in both the 3'-terminal and 5'-terminal region of a constituent DsiRNA agent or in some instances in various positions within the sequence. With the restrictions noted above in mind, any number and combination of modifications can be incorporated into the constituent DsiRNA agent(s). Where multiple modifications are present, they may be the same or different. Modifications to bases, sugar moieties, the phosphate backbone, and their combinations are contemplated. Either 5'-terminus of a constituent DsiRNA agent can be phosphorylated.

[0179] Examples of modifications contemplated for the phosphate backbone of the bifunctional or functional group-tethered DsiRNA agents of the instant invention include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester modifications such as alkylphosphotriesters, and the like. Examples of modifications contemplated for the sugar moiety include 2'-alkyl pyrimidine, such as 2'-O-methyl, 2'-fluoro, amino, and deoxy modifications and the like (see, e.g., Amarzguioui et al., 2003). Examples of modifications contemplated for the base groups include abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's, could also be incorporated. Many other modifications are known and can be used so long as the above criteria are satisfied. Examples of modifications are also disclosed in U.S. Pat. Nos. 5,684,143, 5,858,988 and 6,291,438 and in U.S. published patent application No. 2004/0203145 A1. Other modifications are disclosed in Herdewijn (2000), Eckstein (2000), Rusckowski et al. (2000), Stein et al. (2001); Vorobjev et al. (2001).

[0180] One or more modifications contemplated can be incorporated into either strand. The placement of the modifications in the constituent DsiRNA agent(s) of the bifunctional or functional group-tethered DsiRNA agents of the invention can greatly affect the characteristics of the constituent DsiRNA agent(s), including conferring greater potency and stability, reducing toxicity, enhancing Dicer processing, and minimizing an immune response. In one embodiment, the antisense strand or the sense strand or both strands have one or more 2'-O-methyl modified nucleotides. In another embodiment, the antisense strand contains 2'-O-methyl modified nucleotides. In another embodiment, the antisense stand contains a 3' overhang that is comprised of 2'-O-methyl modified nucleotides. The antisense strand could also include additional 2'-O-methyl modified nucleotides.

[0181] In certain embodiments of the present invention, the constituent DsiRNA(s) of the bifunctional or functional group-tethered DsiRNA agents of the invention possess one or more properties which enhance constituent DsiRNA processing by Dicer. According to these embodiments, the constituent DsiRNA agent has a length sufficient such that it is processed by Dicer to produce an active siRNA and at least one of the following properties: (i) the constituent DsiRNA agent is asymmetric, e.g., has a 3' overhang on the antisense strand and (ii) the constituent DsiRNA agent has a modified 3' end on the sense strand to direct orientation of Dicer binding and processing of the dsRNA region to an active siRNA. In certain such embodiments, the presence of one or more base paired deoxyribonucleotides in a region of the sense strand that is 3' to the projected site of Dicer enzyme cleavage and corresponding region of the antisense strand that is 5' of the projected site of Dicer enzyme cleavage can also serve to orient such a constituent DsiRNA molecule for appropriate directionality of Dicer enzyme cleavage.

[0182] In certain embodiments, the length of such a dsDNA region (or length of the region comprising DNA:DNA base pairs) is 1-50 base pairs, optionally 2-30 base pairs, preferably 2-20 base pairs, and more preferably 2-10 base pairs. Thus, a DNA:DNA-extended constituent DsiRNA of the instant invention may possess a dsDNA region that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more base pairs in length.

[0183] In some embodiments, the longest strand in a constituent DsiRNA of the bifunctional or functional group-tethered DsiRNA agent of the invention comprises 29-43 nucleotides. In one embodiment, the constituent DsiRNA agent is asymmetric such that the 3' end of the sense strand and 5' end of the antisense strand form a blunt end, and the 3' end of the antisense strand overhangs the 5' end of the sense strand. In certain embodiments, the 3' overhang of the antisense strand is 1-10 nucleotides, and optionally is 1-4 nucleotides, for example 2 nucleotides. Both the sense and the antisense strand may also have a 5' phosphate.

[0184] In certain embodiments, the sense strand of a constituent DsiRNA of the bifunctional or functional group-tethered DsiRNA agent of the invention that comprises base paired deoxyribonucleotide residues has a total length of between 26 nucleotides and 39 nucleotides. In certain embodiments, the length of the sense strand is between 27 and 35 nucleotides, or, optionally, is between 27 and 33 nucleotides in length. Optionally, the sense strand has a length of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 nucleotides. In related embodiments, the antisense strand has a length of between 27 and 43 nucleotides in length. In certain such embodiments, the antisense strand has a length of between 27 and 39 nucleotides in length, of between 27 and 35 nucleotides in length, of between 28 and 37 nucleotides in length, or, optionally, of between 29 and 35 nucleotides in length. Optionally, the antisense strand has a length of 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43 nucleotides.

[0185] In certain embodiments, the presence of one or more base paired deoxyribonucleotides in a region of the sense strand that is 3' of the projected site of Dicer enzyme cleavage and corresponding region of the antisense strand that is 5' of the projected site of Dicer enzyme cleavage within a constituent DsiRNA can serve to direct Dicer enzyme cleavage of such a molecule. While certain constituent DsiRNA agents can possess a sense strand deoxyribonucleotide that is located at position 24 or more 3' when counting from position 1 at the 5' end of the sense strand, and having this position 24 or more 3' deoxyribonucleotide of the sense strand base pairing with a cognate deoxyribonucleotide of the antisense strand, in some embodiments, it is also possible to direct Dicer to cleave a shorter product, e.g., a 19mer or a 20mer via inclusion of deoxyribonucleotide residues at, e.g., position 20 of the sense strand. Such a position 20 deoxyribonucleotide base pairs with a corresponding deoxyribonucleotide of the antisense strand, thereby directing Dicer-mediated excision of a 19mer as the most prevalent Dicer product (it is noted that the antisense strand can also comprise one or two deoxyribonucleotide residues immediately 3' of the antisense residue that base pairs with the position 20 deoxyribonucleotide residue of the sense strand in such embodiments, to further direct Dicer cleavage of the antisense strand). In such embodiments, the double-stranded DNA region (which is inclusive of modified nucleic acids that block Dicer cleavage) will generally possess a length of greater than 1 or 2 base pairs (e.g., 3 to 5 base pairs or more), in order to direct Dicer cleavage to generate what is normally a non-preferred length of Dicer cleavage product. A parallel approach can also be taken to direct Dicer excision of 20mer siRNAs from constituent DsiRNA(s) of the bifunctional or functional group-tethered DsiRNA agents of the invention, with the positioning of the first deoxyribonucleotide residue of the sense strand (when surveying the sense strand from position 1 at the 5' terminus of the sense strand of a constituent DsiRNA) occurring at position 21.

[0186] In certain embodiments, the sense strand of the constituent DsiRNA of the bifunctional or functional group-tethered DsiRNA agent of the invention is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the constituent DsiRNA agent is designed to direct orientation of Dicer binding and processing via sense strand modification. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the DsiRNA agent to direct the orientation of Dicer processing of the antisense strand. In a further embodiment of the present invention, two terminal DNA bases are substituted for two ribonucleotides on the 3'-end of the sense strand forming a blunt end of the duplex on the 3' end of the sense strand and the 5' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the antisense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end. In certain embodiments of the instant invention, the modified nucleotides (e.g., deoxyribonucleotides) of the penultimate and ultimate positions of the 3' terminus of the sense strand base pair with corresponding modified nucleotides (e.g., deoxyribonucleotides) of the antisense strand (optionally, the penultimate and ultimate residues of the 5' end of the antisense strand in those constituent DsiRNA agents of the instant invention possessing a blunt end at the 3' terminus of the sense strand/5' terminus of the antisense strand).

[0187] The sense and antisense strands of a constituent(s) DsiRNA of the bifunctional or functional group-tethered DsiRNA agent of the instant invention anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the constituent DsiRNA agent has a sequence length of at least 19 nucleotides, wherein these nucleotides are in the 21-nucleotide region adjacent to the 3' end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to anneal with and/or decrease levels of such a target RNA.

[0188] The constituent DsiRNA(s) of the bifunctional or functional group-tethered DsiRNA agent of the instant invention may possess one or more deoxyribonucleotide base pairs located at any positions of sense and antisense strands that are located 3' of the projected Dicer cleavage site of the sense strand and 5' of the projected Dicer cleavage site of the antisense strand. In certain embodiments, one, two, three or all four of positions 24-27 of the sense strand (starting from position 1 at the 5' terminus of the sense strand) are deoxyribonucleotides, each deoxyribonucleotide of which base pairs with a corresponding deoxyribonucleotide of the antisense strand. In certain embodiments, the deoxyribonucleotides of the 5' region of the antisense strand (e.g., the region of the antisense strand located 5' of the projected Dicer cleavage site for a given constituent DsiRNA molecule) are not complementary to the target RNA to which the constituent DsiRNA agent is directed. In related embodiments, the entire region of the antisense strand located 5' of the projected Dicer cleavage site of a constituent DsiRNA agent is not complementary to the target RNA to which the constituent DsiRNA agent is directed. In certain embodiments, the deoxyribonucleotides of the antisense strand or the entire region of the antisense strand that is located 5' of the projected Dicer cleavage site of the constituent DsiRNA agent is not sufficiently complementary to the target RNA to enhance annealing of the antisense strand of the constituent DsiRNA to the target RNA when the antisense strand is annealed to the target RNA under conditions sufficient to allow for annealing between the antisense strand and the target RNA (e.g., a "core" antisense strand sequence lacking the DNA-extended region anneals equally well to the target RNA as the same "core" antisense strand sequence also extended with sequence of the DNA-extended region, optionally also comprising residual sequence from the RNase H-cleaved domain post-RNase H processing).

[0189] A constituent DsiRNA of a bifunctional or functional group-tethered DsiRNA agent may also have one or more of the following additional properties: (a) the antisense strand has a right shift from the typical 21mer, (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings and (c) base modifications such as locked nucleic acid(s) may be included in the 5' end of the sense strand. A "typical" 21mer siRNA is designed using conventional techniques. In one technique, a variety of sites are commonly tested in parallel or pools containing several distinct siRNA duplexes specific to the same target with the hope that one of the reagents will be effective (Ji et al., 2003). Other techniques use design rules and algorithms to increase the likelihood of obtaining active RNAi effector molecules (Schwarz et al., 2003; Khvorova et al., 2003; Ui-Tei et al., 2004; Reynolds et al., 2004; Krol et al., 2004; Yuan et al., 2004; Boese et al., 2005). High throughput selection of siRNA has also been developed (U.S. published patent application No. 2005/0042641 A1). Potential target sites can also be analyzed by secondary structure predictions (Heale et al., 2005). This 21mer is then used to design a right shift to include 3-9 additional nucleotides on the 5' end of the 21mer. The sequence of these additional nucleotides may have any sequence. In one embodiment, the added ribonucleotides are based on the sequence of the target gene. Even in this embodiment, full complementarity between the target sequence and the antisense siRNA is not required.

[0190] The first and second oligonucleotides of a bifunctional or functional group-tethered DsiRNA agent of the instant invention are not required to be completely complementary. They only need to be substantially complementary to anneal under biological conditions and to provide a substrate for RNase H and, in turn, for Dicer that produces siRNA(s) sufficiently complementary to target sequence(s). Locked nucleic acids, or LNA's, are well known to a skilled artisan (Elman et al., 2005; Kurreck et al., 2002; Crinelli et al., 2002; Braasch and Corey, 2001; Bondensgaard et al., 2000; Wahlestedt et al., 2000). In one embodiment, an LNA is incorporated at the 5' terminus of the sense strand of a constituent DsiRNA agent. In another embodiment, an LNA is incorporated at the 5' terminus of the sense strand in duplexes designed to include a 3' overhang on the antisense strand (within a constituent DsiRNA agent of the bifunctional or functional group-tethered DsiRNA agent of the instant invention).

[0191] In certain embodiments, the constituent DsiRNA of a bifunctional or functional group-tethered DsiRNA agent of the instant invention has an asymmetric structure, with the sense strand having a 27-base pair length, and the antisense strand having a 29-base pair length with a 2 base 3'-overhang. Such constituent agents optionally may possess between one and four deoxyribonucleotides of the 3' terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In other embodiments, the sense strand has a 28-base pair length, and the antisense strand has a 30-base pair length with a 2 base 3'-overhang. Such agents optionally may possess between one and five deoxyribonucleotides of the 3' terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In additional embodiments, the sense strand has a 29-base pair length, and the antisense strand has a 31-base pair length with a 2 base 3'-overhang. Such agents optionally possess between one and six deoxyribonucleotides of the 3' terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In further embodiments, the sense strand has a 30-base pair length, and the antisense strand has a 32-base pair length with a 2 base 3'-overhang. Such agents optionally possess between one and seven deoxyribonucleotides of the 3' terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In other embodiments, the sense strand has a 31-base pair length, and the antisense strand has a 33-base pair length with a 2 base 3'-overhang. Such agents optionally possess between one and eight deoxyribonucleotides of the 3' terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In additional embodiments, the sense strand has a 32-base pair length, and the antisense strand has a 34-base pair length with a 2 base 3'-overhang. Such agents optionally possess between one and nine deoxyribonucleotides of the 3' terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In certain further embodiments, the sense strand has a 33-base pair length, and the antisense strand has a 35-base pair length with a 2 base 3'-overhang. Such agents optionally possess between one and ten deoxyribonucleotides of the 3' terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In still other embodiments, any of these constituent DsiRNA agents have an asymmetric structure that further contains 2 deoxyribonucleotides at the 3' end of the sense strand in place of two of the ribonucleotides; optionally, these 2 deoxyribonucleotides base pair with cognate deoxyribonucleotides of the antisense strand.

[0192] Certain bifunctional or functional group-tethered DsiRNA agents containing two separate oligonucleotides can be linked by a third structure. The third structure will not block Dicer activity on the constituent DsiRNA agent and will not interfere with the directed destruction of the RNA transcribed from the target gene. In one embodiment, the third structure may be a chemical linking group. Many suitable chemical linking groups are known in the art and can be used. Alternatively, the third structure may be an oligonucleotide that links the two oligonucleotides of the bifunctional or functional group-tethered DsiRNA agent in a manner such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the double stranded nucleic acid composition. The hairpin structure will not block Dicer activity on a constituent DsiRNA agent and will not interfere with the directed destruction of target RNA(s) by such constituent DsiRNA agent(s).

[0193] In certain embodiments, the constituent DsiRNA agent(s) of the constituent of the invention has several properties which enhance processing of constituent DsiRNA agents by Dicer. According to such embodiments, the constituent DsiRNA agent has a length sufficient such that it is processed by Dicer to produce an siRNA and at least one of the following properties: (i) the constituent DsiRNA agent is asymmetric, e.g., has a 3' overhang on the sense strand and (ii) the constituent DsiRNA agent has a modified 3' end on the antisense strand to direct orientation of Dicer binding and processing of the dsRNA region to an active siRNA. According to these embodiments, the longest strand in the constituent DsiRNA agent comprises 25-43 nucleotides. In one embodiment, the sense strand comprises 25-39 nucleotides and the antisense strand comprises 26-43 nucleotides. The resulting double stranded nucleic acid can have an overhang on the 3' end of the sense strand. The overhang is 1-4 nucleotides, such as 2 nucleotides. The antisense or sense strand may also have a 5' phosphate.

[0194] In certain embodiments, the sense strand of a constituent DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the constituent DsiRNA agent is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the double stranded nucleic acid to direct the orientation of Dicer processing. In a further embodiment, two terminal DNA bases are located on the 3' end of the sense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the antisense strand and the 3' end of the sense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the antisense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

[0195] In certain other embodiments, the antisense strand of a constituent DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the antisense strand, i.e., the constituent DsiRNA agent is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the antisense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the double stranded nucleic acid to direct the orientation of Dicer processing. In a further invention, two terminal DNA bases are located on the 3' end of the antisense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the sense strand and the 3' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the sense strand. This is also an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

[0196] The sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the double stranded nucleic acid has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3' end of antisense strand and are sufficiently complementary to a nucleotide sequence of the target RNA to direct RNA interference.

[0197] Additionally, a constituent DsiRNA agent structure can be optimized to ensure that the oligonucleotide segment generated from Dicer's cleavage will be the portion of the oligonucleotide that is most effective in inhibiting gene expression. For example, in one embodiment, a 27-35-bp oligonucleotide of the constituent DsiRNA agent structure is synthesized wherein the anticipated 21 to 22-bp segment that will inhibit gene expression is located on the 3'-end of the antisense strand. The remaining bases located on the 5'-end of the antisense strand will be cleaved by Dicer and will be discarded. This cleaved portion can be homologous (i.e., based on the sequence of the target sequence) or non-homologous and added to extend the nucleic acid strand. Such extension can be performed with base paired DNA residues (double stranded DNA:DNA extensions), resulting in extended constituent DsiRNA agents having improved efficacy or duration of effect than corresponding double stranded RNA:RNA-extended constituent DsiRNA agents. Indeed, in certain embodiments, such regions of DNA:DNA extension can be used as sequences that join two otherwise independent DsiRNA moieties into a single bifunctional agent (such as the agent shown in FIG. 4).

[0198] US 2007/0265220 discloses that 27mer DsiRNAs show improved stability in serum over comparable 21mer siRNA compositions, even absent chemical modification. Modifications of constituent DsiRNA agents, such as inclusion of 2'-O-methyl RNA in the antisense strand, in patterns such as detailed in US 2007/0265220 and otherwise herein, when coupled with addition of a 5' Phosphate, can improve stability of constituent DsiRNA agents. Addition of 5'-phosphate to all strands in synthetic RNA duplexes may be an inexpensive and physiological method to confer some limited degree of nuclease stability.

[0199] The chemical modification patterns of the constituent DsiRNA agents of the instant invention are designed to enhance the efficacy of such agents. Accordingly, such modifications are designed to avoid reducing potency of constituent DsiRNA agents; to avoid interfering with Dicer processing of constituent DsiRNA agents; to improve stability in biological fluids (reduce nuclease sensitivity) of constituent DsiRNA agents; or to block or evade detection by the innate immune system. Such modifications are also designed to avoid being toxic and to avoid increasing the cost or impact the ease of manufacturing the instant bifunctional and functional group-tethered DsiRNA agents of the invention.

Joining of Constituent DsiRNA Agents to Form Bifunctional or Functional Group-Tethered DsiRNA Agents

[0200] Certain bifunctional and functional group-tethered DsiRNA agents of the instant invention comprise an RNase H cleavable domain, with such an RNase H cleavable domain constituting a double-stranded span of nucleotides that joins a constituent DsiRNA either to another constituent DsiRNA or to a functional group. The exemplary RNase H cleavable domain of the invention is a double stranded sequence comprising a sufficient length of RNA:DNA duplexed nucleotides to provoke RNase H cleavage. In certain embodiments, the length of such a double-stranded RNA:DNA RNase H-cleavable region is 4-50 base pairs, optionally 4-30 base pairs, 4-20 base pairs, 4-16 base pairs, 4-10 base pairs, 6-10 base pairs and in certain embodiments 7-9 base pairs. Thus, an RNase H-cleavable agent of the instant invention may possess a double-stranded RNA:DNA RNase H-cleavable region that is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more base pairs in length.

Conjugation and Delivery of Bifunctional and Functional Group-Tethered DsiRNA Agents

[0201] In certain embodiments, the present invention relates to a method for treating a subject having or at risk of developing a disease or disorder. In such embodiments, the DsiRNA can act as a novel therapeutic agent for controlling the disease or disorder. The method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that the expression, level and/or activity a target RNA is reduced. The expression, level and/or activity of a polypeptide endoded by the target RNA might also be reduced by a DsiRNA of the instant invention.

[0202] In the treatment of a disease or disorder, the DsiRNA can be brought into contact with the cells or tissue exhibiting or associated with a disease or disorder. For example, DsiRNA substantially identical to all or part of a target RNA sequence, may be brought into contact with or introduced into a diseased, disease-associated or infected cell, either in vivo or in vitro. Similarly, DsiRNA substantially identical to all or part of a target RNA sequence may administered directly to a subject having or at risk of developing a disease or disorder.

[0203] Therapeutic use of the DsiRNA agents of the instant invention can involve use of formulations of DsiRNA agents comprising multiple different DsiRNA agent sequences. For example, two or more, three or more, four or more, five or more, etc. of the presently described agents can be combined to produce a formulation that, e.g., targets multiple different regions of one or more target RNA(s). A DsiRNA agent of the instant invention may also be constructed such that either strand of the DsiRNA agent independently targets two or more regions of a target RNA. Use of multifunctional DsiRNA molecules that target more then one region of a target nucleic acid molecule is expected to provide potent inhibition of RNA levels and expression. For example, a single multifunctional DsiRNA construct of the invention can target both conserved and variable regions of a target nucleic acid molecule, thereby allowing down regulation or inhibition of, e.g., different strain variants of a virus, or splice variants encoded by a single target gene.

[0204] A DsiRNA agent of the invention can be conjugated (e.g., at its 5' or 3' terminus of its sense or antisense strand) or unconjugated to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye, cholesterol, or the like). Modifying DsiRNA agents in this way may improve cellular uptake or enhance cellular targeting activities of the resulting DsiRNA agent derivative as compared to the corresponding unconjugated DsiRNA agent, are useful for tracing the DsiRNA agent derivative in the cell, or improve the stability of the DsiRNA agent derivative compared to the corresponding unconjugated DsiRNA agent.

Enhanced In Vivo Efficacy and Duration of Effect of Bifunctional DsiRNA Agents

[0205] In certain embodiments, the bifunctional agents of the invention (both RNase H-cleavable bifunctional agents and dsDNA extension sequence-joined bifunctional agents) can exhibit enhanced in vivo efficacy, enhanced in vivo duration of effect, or both, as compared to a tandem siRNA agent directed against the same target RNA sequence(s) (e.g., tandem siRNA agents in which 19-21mer siRNA moieties are joined by an RNase H-cleavable sequence). In vivo assessment of the extent of inhibition of target RNA(s) by such bifunctional agents can be assessed either phenotypically (e.g., via assessment of therapeutic effect following administration of such an agent to a subject) or via direct assessment of expression levels of target RNAs and/or encoded target protein(s), e.g., via art-recognized methods of expression level assessment, including Northern blot or other hybridization-based detection (e.g., array-based expression profiling), RT-PCR (qRT-PCR), ELISA, Western blot, etc. The extent to which a bifunctional agent of the invention inhibits expression of a target RNA can be assessed relative to an untreated control, e.g., a bifunctional agent of the invention can be shown to reduce a target RNA (or multiple target RNAs) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more than in an untreated subject (and/or cell). Additionally or alternatively, the extent to which a bifunctional agent of the invention inhibits the level of a target RNA(s) can be assessed relative to a corresponding 19-21mer tandem siRNA agent (e.g., an agent in which 19-21mer siRNA moieties are joined by an RNase H-cleavable sequence). In such embodiments, the bifunctional DsiRNA agents of the invention are identified to exhibit at least 10% greater levels of inhibition of a target RNA(s) than a corresponding tandem siRNA. In related embodiments, the bifunctional DsiRNA agents of the invention are identified to exhibit at least 20% greater levels of inhibition of a target RNA(s), at least 30% greater levels of inhibition of a target RNA(s), at least 40% greater levels of inhibition of a target RNA(s), at least 50% greater levels of inhibition of a target RNA(s), at least 60% greater levels of inhibition of a target RNA(s), at least 70% greater levels of inhibition of a target RNA(s), at least 80% greater levels of inhibition of a target RNA(s), at least 90% greater levels of inhibition of a target RNA(s) or at least 95% greater levels of inhibition of a target RNA(s) than a corresponding tandem siRNA. In certain other embodiments, the bifunctional DsiRNA agents of the invention are identified to exhibit at least 1.5-fold greater inhibition of target RNA level(s) than a corresponding tandem siRNA. In a related embodiment, the bifunctional DsiRNA agents of the invention are identified to exhibit at least two-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 10-fold greater, or at least 20-fold greater inhibition of target RNA level(s) than a corresponding tandem siRNA (e.g., administered at the same concentration as the bifunctional DsiRNA of the invention).

[0206] In vivo assessment of the duration of inhibition of target RNA(s) by bifunctional agents of the invention can also be assessed either via phenotypic evaluation (e.g., via assessment of therapeutic effect at a specified time/over a time course following administration of such an agent to a subject) or via direct assessment of expression levels of target RNAs and/or encoded target protein(s) at a specified time/over a time course following administration of a bifunctional DsiRNA agent of the invention, e.g., via art-recognized methods of expression level assessment, including Northern blot or other hybridization-based detection (e.g., array-based expression profiling), RT-PCR (qRT-PCR), ELISA, Western blot, etc. The duration of time for which a bifunctional agent of the invention inhibits expression of a target RNA, (e.g., the duration of time over which target RNA levels are reduced by greater than a specified amount, e.g., greater than 10% reduction, greater than 20% reduction, greater than 30% reduction, greater than 40% reduction, greater than 50% reduction, greater than 60% reduction, greater than 70% reduction, greater than 80% reduction, greater than 90% reduction, or more) can be assessed relative to an untreated control level, e.g., a bifunctional agent of the invention can be shown to reduce a target RNA (or multiple target RNAs) by a stated percentage for a duration of at least 24 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, or at least 20 days or more in a subject (and/or cell). Additionally or alternatively, the duration of time for which a bifunctional agent of the invention inhibits the level of a target RNA(s) can be assessed relative to a corresponding 19-21mer tandem siRNA agent (e.g., an agent in which 19-21mer siRNA moieties are joined by an RNase H-cleavable sequence). In such embodiments, the bifunctional DsiRNA agents of the invention are identified to reduce a target RNA (or multiple target RNAs) by a stated percentage for a duration of at least 24 hours more, at least 48 hours more, at least 3 days more, at least 4 days more, at least 5 days more, at least 6 days more, at least 7 days more, at least 8 days more, at least 9 days more, at least 10 days more, at least 12 days more, at least 14 days more, at least 16 days more, at least 18 days more, or at least 20 days more in a subject (and/or cell) than a corresponding tandem siRNA. In related embodiments, the bifunctional DsiRNA agents of the invention are identified to exhibit at least 20% longer duration of inhibition of a target RNA(s) to a given percent or fold reduction, at least 30% longer duration of inhibition of a target RNA(s) to a given percent or fold reduction, at least 40% longer duration of inhibition of a target RNA(s) to a given percent or fold reduction, at least 50% longer duration of inhibition of a target RNA(s) to a given percent or fold reduction, at least 60% longer duration of inhibition of a target RNA(s) to a given percent or fold reduction, at least 70% longer duration of inhibition of a target RNA(s) to a given percent or fold reduction, at least 80% longer duration of inhibition of a target RNA(s) to a given percent or fold reduction, at least 90% longer duration of inhibition of a target RNA(s) to a given percent or fold reduction or at least 95% longer duration of inhibition of a target RNA(s) to a given percent or fold reduction than a corresponding tandem siRNA. In certain other embodiments, the bifunctional DsiRNA agents of the invention are identified to exhibit at least 1.5-fold longer duration of inhibition of target RNA level(s) than a corresponding tandem siRNA. In a related embodiment, the bifunctional DsiRNA agents of the invention are identified to exhibit at least two-fold longer duration of inhibition, at least 3-fold longer duration of inhibition, at least 4-fold longer duration of inhibition, at least 5-fold longer duration of inhibition, at least 10-fold longer duration of inhibition, or at least 20-fold longer duration of inhibition of target RNA level(s) than a corresponding tandem siRNA (e.g., administered at the same concentration as the bifunctional DsiRNA of the invention).

[0207] The therapeutic impact of a bifunctional agent of the invention can also be assessed in evaluating the efficacy of such an agent. In such embodiments, a bifunctional DsiRNA agent of the invention can be shown to exhibit, e.g., at least a 10% reduction in symptoms and/or indicator of disease status relative to an untreated control subject, relative to a subject treated with a tandem siRNA agent (e.g., an agent in which 19-21mer siRNA moieties are joined by an RNase H-cleavable sequence), or relative to appropriate control. In related embodiments, a bifunctional DsiRNA agent of the invention can be shown to exhibit, e.g., at least a 20% reduction in symptoms and/or indicator of disease status, at least a 30% reduction in symptoms and/or indicator of disease status, at least a 40% reduction in symptoms and/or indicator of disease status, at least a 50% reduction in symptoms and/or indicator of disease status, at least a 60% reduction in symptoms and/or indicator of disease status, at least a 70% reduction in symptoms and/or indicator of disease status, at least a 80% reduction in symptoms and/or indicator of disease status, at least a 90% reduction in symptoms and/or indicator of disease status, at least a 95% reduction in symptoms and/or indicator of disease status, or a complete reduction in symptoms and/or indicator of disease status, relative to an untreated control subject, relative to a subject treated with a tandem siRNA agent, or relative to other appropriate control.

[0208] One advantage of DsiRNA agents, including the bifunctional DsiRNA agents of the invention, is their ability to act as effective inhibitor agents at concentrations in the environment of a cell of 1 nanomolar or less (e.g., efficacy at approximately 500 pM or less, efficacy at approximately 200 pM or less, efficacy at approximately 100 pM or less, efficacy at approximately 50 pM or less, efficacy at about 20 pM or less, or efficacy at about 10 pM or less). Given the observed potency of DsiRNA agents, as well as the duration of effects identified for such agents, the bifunctional DsiRNA agents of the invention can be administered at therapeutically effective doses to a subject that are lower than those which would correspondingly be necessary for a tandem 19-21mer siRNA agent to be therapeutically effective. With the caveat that dosage ranges for a bifunctional DsiRNA agent of the invention will vary depending upon variables such as mode of delivery, nature of delivery vehicle, etc., exemplary dosage ranges for the bifunctional DsiRNA agents of the invention include 0.005 to 5.0 mg of bifunctional DsiRNA agent per kilogram of body weight of a subject, including, e.g., dosage ranges of 0.05 to 2.0 mg/kg per dose administered as a therapeutically effective dose to a subject.

[0209] In certain embodiments, the bifunctional DsiRNA agents of the invention exhibit enhanced therapeutic efficacy because the delivery of conjoined DsiRNA moieties in a single molecule achieves equivalent dosage and distribution of such DsiRNA moieties, relative to the dosing and distribution that could be achieved via administration of independent DsiRNA moieties to a subject. As such, the bifunctional DsiRNA agents of the invention can also be assessed for enhanced therapeutic efficacy when structured as a bifunctional DsiRNA, as compared to independent DsiRNA moieties (e.g., administered separately). The synergistic impact of packaging DsiRNA agents within such bifunctional structures can be assessed in a subject via evaluation of phenotype and/or therapeutic outcome, e.g., bifunctional DsiRNA agents are 20% more effective (or 30% more effective, 40% more effective, 50% more effective, 60% more effective, 70% more effective, 80% more effective, 90% more effective or 95% more effective) and/or exhibit 1.5-fold or greater duration of effect (or two-fold or greater duration of effect, 3-fold or greater duration of effect, 4-fold or greater duration of effect, 5-fold or greater duration of effect, 10-fold or greater duration of effect or 20-fold or greater duration of effect) upon phenotype, therapeutic outcome and/or RNA levels than corresponding, independent DsiRNA agents directed against the same target RNA(s).

RNAi In Vitro Assay to Assess DsiRNA Agent Activity

[0210] An in vitro assay that recapitulates RNAi in a cell-free system can optionally be used to evaluate DsiRNA-containing constructs. For example, such an assay comprises a 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 DsiRNA agents directed against 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 plasmid using T7 RNA polymerase or via chemical synthesis. Sense and antisense DsiRNA 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.degree. C. followed by 1 hour at 37.degree. 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 .mu.M final concentration), and 10% [vol/vol] lysis buffer containing DsiRNA (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.degree. C. for 10 minutes before adding RNA, then incubated at 25.degree. C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25.times. 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 DsiRNA is omitted from the reaction.

[0211] 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.RTM. (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without DsiRNA and the cleavage products generated by the assay.

Bifunctional Agent Targets

[0212] The bifunctional agents of the invention can target two regions of the same target RNA, or can target two independent sites within two or more target RNAs. As will be appreciated by the skilled artisan, it will be desirable in certain experimental, clinical or therapeutic settings to ensure equivalent levels of delivery of two active agents via use of a single bifunctional molecule such as the ones disclosed herein. Specific pairs of, e.g., oncogenes, growth regulation genes, ligand/receptor pairs (e.g., VEGF/VEGFR, EGF/EGFR, etc.), genes of autocrine loops, angiogenesis factors, etc. can advantageously be simultaneously targeted with the bifunctional agents of the instant invention.

Methods of Introducing Nucleic Acids, Vectors, and Host Cells

[0213] Bifunctional and functional group-tethered DsiRNA agents of the invention may be directly introduced into a cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.

[0214] The bifunctional and functional group-tethered DsiRNA agents of the invention can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like. The nucleic acid may be introduced along with other components that perform one or more of the following activities: enhance nucleic acid uptake by the cell or other-wise increase inhibition of the target RNA.

[0215] A cell having a target RNA may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands.

[0216] Depending on the particular target RNA sequence and the dose of DsiRNA agent material delivered, this process may provide partial or complete loss of function for the target RNA. A reduction or loss of RNA levels or expression (either RNA expression or encoded polypeptide expression) in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary. Inhibition of target RNA levels or expression refers to the absence (or observable decrease) in the level of RNA or RNA-encoded protein. Specificity refers to the ability to inhibit the target RNA without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). Inhibition of target RNA sequence(s) by the bifunctional and functional group-tethered DsiRNA agents of the invention also can be measured based upon the effect of administration of such bifunctional and functional group-tethered DsiRNA agents upon measurable phenotypes such as tumor size for cancer treatment, viral load/titer for viral infectious diseases, etc. either in vivo or in vitro. For viral infectious diseases, reductions in viral load or titer can include reductions of, e.g., 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, and are often measured in logarithmic terms, e.g., 10-fold, 100-fold, 1000-fold, 10.sup.5-fold, 10.sup.6-fold, 10.sup.7-fold reduction in viral load or titer can be achieved via administration of the bifunctional and functional group-tethered DsiRNA agents of the invention to cells, a tissue, or a subject.

[0217] For RNAi-mediated inhibition in a cell line or whole organism, expression of a reporter or drug resistance gene whose protein product is easily assayed can be measured. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present invention.

[0218] Lower doses of injected material and longer times after administration of RNA silencing agent may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells). Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target RNA or translation of target protein. As an example, the efficiency of inhibition may be determined by assessing the amount of gene product in the cell; RNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory DsiRNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.

[0219] The bifunctional or functional group-tethered DsiRNA agent of the invention may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of material may yield more effective inhibition; lower doses may also be useful for specific applications.

RNA Interference Based Therapy

[0220] As is known, RNAi methods are applicable to a wide variety of genes in a wide variety of organisms and the disclosed compositions and methods can be utilized in each of these contexts. Examples of genes which can be targeted by the disclosed compositions and methods include endogenous genes which are genes that are native to the cell or to genes that are not normally native to the cell. Without limitation, these genes include oncogenes, cytokine genes, idiotype (Id) protein genes, prion genes, genes that expresses molecules that induce angiogenesis, genes for adhesion molecules, cell surface receptors, proteins involved in metastasis, proteases, apoptosis genes, cell cycle control genes, genes that express EGF and the EGF receptor, multi-drug resistance genes, such as the MDR1 gene.

[0221] More specifically, a target mRNA of the invention can specify the amino acid sequence of a cellular protein (e.g., a nuclear, cytoplasmic, transmembrane, or membrane-associated protein). In another embodiment, the target mRNA of the invention can specify the amino acid sequence of an extracellular protein (e.g., an extracellular matrix protein or secreted protein). As used herein, the phrase "specifies the amino acid sequence" of a protein means that the mRNA sequence is translated into the amino acid sequence according to the rules of the genetic code. The following classes of proteins are listed for illustrative purposes: developmental proteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, K-RAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53, and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextriinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hernicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases).

[0222] In one aspect, the target mRNA molecule(s) of the invention specifies the amino acid sequence of a protein associated with a pathological condition. For example, the protein may be a pathogen-associated protein (e.g., a viral protein involved in immunosuppression of the host, replication of the pathogen, transmission of the pathogen, or maintenance of the infection), or a host protein which facilitates entry of the pathogen into the host, drug metabolism by the pathogen or host, replication or integration of the pathogen's genome, establishment or spread of infection in the host, or assembly of the next generation of pathogen. Pathogens include RNA viruses such as flaviviruses, picornaviruses, rhabdoviruses, filoviruses, retroviruses, including lentiviruses, or DNA viruses such as adenoviruses, poxviruses, herpes viruses, cytomegaloviruses, hepadnaviruses or others. Additional pathogens include bacteria, fungi, helminths, schistosomes and trypanosomes. Other kinds of pathogens can include mammalian transposable elements. Alternatively, the protein may be a tumor-associated protein or an autoimmune disease-associated protein.

[0223] The target gene(s) may be derived from or contained in any organism. The organism may be a plant, animal, protozoa, bacterium, virus or fungus. See, e.g., U.S. Pat. No. 6,506,559, incorporated herein by reference.

Pharmaceutical Compositions

[0224] In certain embodiments, the present invention provides for a pharmaceutical composition comprising the DsiRNA agent of the present invention. The DsiRNA agent sample can be suitably formulated and introduced into the environment of the cell by any means that allows for a sufficient portion of the sample to enter the cell to induce gene silencing, if it is to occur. Many formulations for double stranded nucleic acid are known in the art and can be used so long as the double stranded nucleic acid gains entry to the target cells so that it can act. See, e.g., U.S. published patent application Nos. 2004/0203145 A1 and 2005/0054598 A1. For example, the DsiRNA agent of the instant invention can be formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids. Formulations of DsiRNA agent with cationic lipids can be used to facilitate transfection of the DsiRNA agent into cells. For example, cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (published PCT International Application WO 97/30731), can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer's instructions.

[0225] Such compositions typically include the nucleic acid molecule and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0226] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0227] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0228] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0229] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0230] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0231] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0232] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0233] The compounds can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).

[0234] The compounds can also be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Pat. No. 6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Pat. No. 6,471,996).

[0235] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0236] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0237] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0238] As defined herein, a therapeutically effective amount of a nucleic acid molecule (i.e., an effective dosage) depends on the nucleic acid selected. For instance, if a plasmid encoding a DsiRNA agent is selected, single dose amounts in the range of approximately 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 .mu.g, or 10, 30, 100, or 1000 mg may be administered. In some embodiments, 1-5 g of the compositions can be administered. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[0239] It can be appreciated that the method of introducing DsiRNA agents into the environment of the cell will depend on the type of cell and the make up of its environment. For example, when the cells are found within a liquid, one preferable formulation is with a lipid formulation such as in lipofectamine and the DsiRNA agents can be added directly to the liquid environment of the cells. Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art. When the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable. Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used. In some instances, it may be preferable to formulate DsiRNA agents in a buffer or saline solution and directly inject the formulated DsiRNA agents into cells, as in studies with oocytes. The direct injection of DsiRNA agents duplexes may also be done. For suitable methods of introducing double stranded nucleic acid (e.g., DsiRNA agents), see U.S. published patent application No. 2004/0203145 A1.

[0240] Suitable amounts of a DsiRNA agent must be introduced and these amounts can be empirically determined using standard methods. Typically, effective concentrations of individual DsiRNA agent species in the environment of a cell will be about 50 nanomolar or less, 10 nanomolar or less, or compositions in which concentrations of about 1 nanomolar or less can be used. In another embodiment, methods utilizing a concentration of about 200 picomolar or less, and even a concentration of about 50 picomolar or less, can be used in many circumstances.

[0241] The method can be carried out by addition of the DsiRNA agent compositions to any extracellular matrix in which cells can live provided that the DsiRNA agent composition is formulated so that a sufficient amount of the DsiRNA agent can enter the cell to exert its effect. For example, the method is amenable for use with cells present in a liquid such as a liquid culture or cell growth media, in tissue explants, or in whole organisms, including animals, such as mammals and especially humans.

[0242] The level or activity of a target RNA can be determined by any suitable method now known in the art or that is later developed. It can be appreciated that the method used to measure a target RNA and/or the expression of a target RNA can depend upon the nature of the target RNA. For example, if the target RNA encodes a protein, the term "expression" can refer to a protein or the RNA/transcript derived from the target RNA. In such instances, the expression of a target RNA can be determined by measuring the amount of RNA corresponding to the target RNA or by measuring the amount of that protein. Protein can be measured in protein assays such as by staining or immunoblotting or, if the protein catalyzes a reaction that can be measured, by measuring reaction rates. All such methods are known in the art and can be used. Where target RNA levels are to be measured, any art-recognized methods for detecting RNA levels can be used (e.g., RT-PCR, Northern Blotting, etc.). In targeting viral RNAs with the DsiRNA agents of the instant invention, it is also anticipated that measurement of the efficacy of a DsiRNA agent in reducing levels of a target virus in a subject, tissue, in cells, either in vitro or in vivo, or in cell extracts can also be used to determine the extent of reduction of target viral RNA level(s). Any of the above measurements can be made on cells, cell extracts, tissues, tissue extracts or any other suitable source material.

[0243] The determination of whether the expression of a target RNA has been reduced can be by any suitable method that can reliably detect changes in RNA levels. Typically, the determination is made by introducing into the environment of a cell undigested DsiRNA such that at least a portion of that DsiRNA agent enters the cytoplasm, and then measuring the level of the target RNA. The same measurement is made on identical untreated cells and the results obtained from each measurement are compared.

[0244] The DsiRNA agent can be formulated as a pharmaceutical composition which comprises a pharmacologically effective amount of a DsiRNA agent and pharmaceutically acceptable carrier. A pharmacologically or therapeutically effective amount refers to that amount of a DsiRNA agent effective to produce the intended pharmacological, therapeutic or preventive result. The phrases "pharmacologically effective amount" and "therapeutically effective amount" or simply "effective amount" refer to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 20% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 20% reduction in that parameter.

[0245] Suitably formulated pharmaceutical compositions of this invention can be administered by any means known in the art such as by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In some embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.

[0246] In general, a suitable dosage unit of double stranded nucleic acid will be in the range of 0.001 to 0.25 milligrams per kilogram body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day. Pharmaceutical composition comprising the double stranded nucleic acid can be administered once daily. However, the therapeutic agent may also be dosed in dosage units containing two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In that case, the double stranded nucleic acid contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage unit. The dosage unit can also be compounded for a single dose over several days, e.g., using a conventional sustained release formulation which provides sustained and consistent release of the double stranded nucleic acid over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. Regardless of the formulation, the pharmaceutical composition must contain double stranded nucleic acid in a quantity sufficient to inhibit expression of the target gene in the animal or human being treated. The composition can be compounded in such a way that the sum of the multiple units of double stranded nucleic acid together contain a sufficient dose.

[0247] Data can be obtained from cell culture assays and animal studies to formulate a suitable dosage range for humans. The dosage of compositions of the invention lies within a range of circulating concentrations that include the ED.sub.50 (as determined by known methods) with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels of double stranded nucleic acid in plasma may be measured by standard methods, for example, by high performance liquid chromatography.

[0248] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.

Methods of Treatment

[0249] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease or disorder caused, in whole or in part, by the expression of a target RNA and/or the presence of such target RNA (e.g., in the context of a viral infection, the presence of a target RNA of the viral genome, capsid, host cell component, etc.).

[0250] "Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent (e.g., a DsiRNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.

[0251] In one aspect, the invention provides a method for preventing in a subject, a disease or disorder as described above, by administering to the subject a therapeutic agent (e.g., a DsiRNA agent or vector or transgene encoding same). Subjects at risk for the disease can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the detection of, e.g., viral particles in a subject, or the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

[0252] Another aspect of the invention pertains to methods of treating subjects therapeutically, i.e., alter onset of symptoms of the disease or disorder. These methods can be performed in vitro (e.g., by culturing the cell with the DsiRNA agent) or, alternatively, in vivo (e.g., by administering the DsiRNA agent to a subject).

[0253] With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype"). Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the target RNA molecules of the present invention or target RNA modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0254] Therapeutic agents can be tested in an appropriate animal model. For example, a DsiRNA agent (or expression vector or transgene encoding same) as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with said agent. Alternatively, a therapeutic agent can be used in an animal model to determine the mechanism of action of such an agent. For example, an agent can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent can be used in an animal model to determine the mechanism of action of such an agent.

[0255] The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000).

[0256] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

[0257] The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

Example 1

Methods

Oligonucleotide Synthesis

[0258] Individual RNA strands are synthesized and HPLC purified according to standard methods (Integrated DNA Technologies, Coralville, Iowa). All oligonucleotides are quality control released on the basis of chemical purity by HPLC analysis and full length strand purity by mass spectrometry analysis. Duplex RNA DsiRNAs are prepared before use by mixing equal quantities of each strand, briefly heating to 100.degree. C. in RNA buffer (IDT) and then allowing the mixtures to cool to room temperature.

Cell Culture and RNA Transfection

[0259] HeLa cells are obtained from ATCC and maintained in Dulbecco's modified Eagle medium (HyClone) supplemented with 10% fetal bovine serum (HyClone) at 37.degree. C. under 5% CO.sub.2. For RNA transfections, HeLa cells are seeded overnight in 6-well plates at a density of 4.times.10.sup.5 cells/well in a final volume of 2 mL. 24 hours later, cells are transfected with the DsiRNA duplexes as specified at a final concentration of 10 pM, 100 pM, 1 nM, 10 nM or 20 nM using Oligofectamine.TM. (Invitrogen) and following the manufacturer's instructions. For 20 nM transfections, 84, of a 5 .mu.M stock solution of each bifunctional or functional group-tethered DsiRNA is mixed with 200 .mu.L of Opti-MEM I (Invitrogen). In a separate tube, 12 .mu.L of Oligofectamine is mixed with 484, of Opti-MEM I. After a 5 minute incubation at room temperature (RT) the bifunctional or functional group-tethered DsiRNA and Oligofectamine aliquots are combined, gently vortexed, and further incubated for 20 minutes at RT to allow bifunctional or functional group-tethered DsiRNA:Oligofectamine complexes (transfection mixes) to form. Finally, culture medium is added to bring each transfection mix to a final volume of 2 mL. After a 6 hour incubation, the transfection/culture medium in each well is replaced with fresh culture medium and cells are incubated for an additional 18 hours.

RNA Isolation and Analysis

[0260] Cells are washed once with 2 mL of PBS, and total RNA is extracted using RNeasy Mini Kit.TM. (Qiagen) and eluted in a final volume of 30 .mu.L. 1 .mu.g of total RNA is reverse-transcribed using Transcriptor 1.sup.st Strand cDNA Kit.TM. (Roche) and random hexamers following manufacturer's instructions. One-thirtieth (0.66 .mu.L) of the resulting cDNA is mixed with 54, of IQ Multiplex Powermix (Bio-Rad) together with 3.33 .mu.L of H.sub.2O and 1 .mu.L of a 3 .mu.M mix containing 2 sets of primers and probes specific for human genes that are assayed (e.g., K-RAS, HPRT1, etc.).

Quantitative RT-PCR

[0261] A CFX96 Real-time System with a C1000 Thermal cycler (Bio-Rad) is used for the amplification reactions. PCR conditions are: 95.degree. C. for 3 min; and then cycling at 95.degree. C., 10 sec; 55.degree. C., 1 min for 40 cycles. Each sample is tested in triplicate. Relative test mRNA levels are compared with mRNA levels obtained in control samples treated with the transfection reagent plus a control mismatch duplex, or untreated. Data is analyzed using Bio-Rad CFX Manager version 1.0 software.

Example 2

Efficacy of RNase H-Cleavable Bifunctional DsiRNA Agents

[0262] Bifunctional DsiRNA agents possessing RNase H-cleavable joining sequences are examined for efficacy of sequence-specific target mRNA inhibition. Specifically, bifunctional DsiRNA agents possessing RNase H-cleavable joining sequences and targeting HPRT1 and K-RAS targets with sequences as shown in FIG. 1 and FIG. 2 are transfected into HeLa cells at a fixed concentration of 10 pM, 100 pM, 1 nM, 10 nM or 20 nM and HPRT1 and K-RAS expression levels are measured 24 hours later. Transfections are performed in duplicate, and each duplicate is assayed in triplicate for HPRT1 and K-RAS expression by qPCR. Under these conditions (10 pM, 100 pM, 1 nM, 10 nM or 20 nM duplexes, Oligofectamine transfection), HPRT1 and K-RAS gene expression is reduced.

Example 3

Efficacy of RNase H-Cleavable Functional Group-Tethered DsiRNA Agents

[0263] Functional group-tethered DsiRNA agents possessing RNase H-cleavable joining sequences are examined for efficacy of sequence-specific target mRNA inhibition and for efficacy of functional group activity. Specifically, functional group-tethered DsiRNA agents possessing RNase H-cleavable joining sequences and as shown in FIG. 3 are synthesized and transfected into HeLa cells at a fixed concentration of 10 pM, 100 pM, 1 nM, 10 nM or 20 nM and HPRT1 expression levels are measured 24 hours later. Transfections are performed in duplicate, and each duplicate is assayed in triplicate for HPRT1 expression by qPCR. Under these conditions (10 pM, 100 pM, 1 nM, 10 nM or 20 nM duplexes, Oligofectamine transfection), HPRT1 gene expression is reduced. Release of the functional group from such agents is also assessed.

Example 4

Efficacy of dsDNA Extended Bifunctional DsiRNA Agents

[0264] Bifunctional DsiRNA agents possessing dsDNA extension joining sequences are examined for efficacy of sequence-specific target mRNA inhibition. Specifically, bifunctional DsiRNA agents possessing dsDNA extension joining sequences and targeting HPRT1 and K-RAS targets with sequences as shown in FIG. 4 are transfected into HeLa cells at a fixed concentration of 10 pM, 100 pM, 1 nM, 10 nM or 20 nM and HPRT1 and K-RAS expression levels are measured 24 hours later. Transfections are performed in duplicate, and each duplicate is assayed in triplicate for HPRT1 and K-RAS expression by qPCR. Under these conditions (10 pM, 100 pM, 1 nM, 10 nM or 20 nM duplexes, Oligofectamine transfection), HPRT1 and K-RAS gene expression is reduced.

Example 5

Enhanced Efficacy of RNase H-Cleavable Bifunctional DsiRNA Agents as Compared to Tandem siRNA Agents

[0265] Bifunctional agents such as those described in Examples 2 and 4 above (and as detailed in FIGS. 1, 2 and 4) are synthesized and transfected into HeLa cells at a fixed concentration of 10 pM, 100 pM, 1 nM or 10 nM. In parallel, tandem 19mer siRNA agents having either RNase H cleavable joining sequences (such as the agent shown at paragraph [0209] of U.S. Patent Application No. 2008/0293655, yet directed against identical target sequences as tested bifunctional DsiRNA sequences (e.g., HPRT1 and K-RAS) or having dsDNA extension joining sequences are synthesized and also transfected into HeLa cells at a fixed concentration of 10 pM, 100 pM, 1 nM or 10 nM. Expression levels are measured at time points of 24 hours later, 2 days later, 4 days later, 6 days later and 10 days later. Transfections are performed in duplicate, and each duplicate is assayed in triplicate for HPRT1 and K-RAS expression by qPCR. Under these conditions (10 pM, 100 pM, 1 nM, 10 nM or 20 nM duplexes, Oligofectamine transfection), HPRT1 and K-RAS gene expression is identified as reduced to a greater extent (showing greater efficacy) and with enhanced duration of effect with bifunctional DsiRNA agents than for corresponding tandem 19mer siRNA agents directed against identical target RNA sequences.

[0266] 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.

[0267] 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.

[0268] 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 DsiRNA molecules with improved RNAi activity.

[0269] 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.

[0270] 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.

[0271] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Sequence CWU 1

1

27113DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1aattcaccgg gga 13214RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 2uccccgguga auuu 14314DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 3aaattcaccg gucg 14414RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 4cgaccgguga auuu 14512DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 5gaaattcacc gg 12656DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 6gccagacuuu guuggauuug aaattcaccg gggagggcuu ucuuugugua uuugcc 56758RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 7ggcaaauaca caaagaaagc ccuccccggu gaauuucaaa uccaacaaag ucuggcgc 58831DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 8gccagacuuu guuggauuug aaattcaccg g 31925RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 9ggagggcuuu cuuuguguau uugcc 251031RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 10ggugaauuuc aaauccaaca aagucuggcg c 311127RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 11ggcaaauaca caaagaaagc ccucccc 271221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 12gccagacuuu guuggauuug a 211321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 13ggagggcuuu cuuuguguau u 211421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 14aaauccaaca aagucuggcg c 211521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 15uacacaaaga aagcccuccc c 211658DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 16gccagacuuu guuggauuug aaattcaccg gucggagggc uuucuuugug uauuugcc 581758RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 17caaauacaca aagaaagccc uccgaccggu gaauuucaaa uccaacaaag ucuggcgc 581825DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 18gccagacuuu guuggauuug aaatt 251933RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 19caccggucgg agggcuuucu uuguguauuu gcc 332027RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 20aauuucaaau ccaacaaagu cuggcgc 272127RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 21caaauacaca aagaaagccc uccgacc 272221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 22gccagacuuu guuggauuug a 212321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 23ggcuuucuuu guguauuugc c 212421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 24aaauccaaca aagucuggcg c 212521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 25caaauacaca aagaaagccc u 212633RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 26ccggugaauu ucaaauccaa caaagucugg cgc 332727RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 27aauuucaaau ccaacaaagu cuggcgc 27

Claims

1. An isolated nucleic acid duplex comprising: a first region comprising first and second oligonucleotide strands comprising ribonucleotides, each strand having 5' and 3' termini, wherein said first region forms a duplex of 23 and 35 ribonucleotides in length; a second region comprising first and second oligonucleotide strands and comprising a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of said second region in an RNase H cleavage assay; and a third region comprising first and second oligonucleotide strands comprising ribonucleotides, each strand having 5' and 3' termini, wherein said third region forms a duplex of 23 and 35 ribonucleotides in length; and wherein said isolated duplex comprises a nick, wherein the position of said nick between immediately adjacent nucleotides is selected from the group consisting of: (a) within said second region in one of said first and second oligonucleotide strands, (b) between said first region and said second region on one strand, and c) between said second region and said third region on one strand.

2. The isolated nucleic acid duplex of claim 1, wherein said second region first oligonucleotide strand comprises deoxynucleotides.

3. The isolated nucleic acid duplex of claim 1, wherein said second region second oligonucleotide strand comprises deoxyribonucleotides.

4. The isolated nucleic acid duplex of claim 2 or 3, wherein said oligonucleotide strand consists of deoxyribonucleotides.

5. The isolated nucleic acid duplex of claim 1, wherein each of said first and third regions, independently, form a duplex of 23 and 30 ribonucleotides in length.

6. The isolated nucleic acid duplex of claim 1, wherein said nick is positioned within said second region on the strand that comprises deoxyribonucleotides.

7. The isolated nucleic acid duplex of claim 1, wherein said nick is positioned between one of said first and second regions or said second and third regions, and wherein said nick is positioned between a deoxyribonucleotide of a second region strand and a ribonucleotide of one of said first or third region strands.

8. The nucleic acid duplex of claim 1, wherein said RNase H cleavage assay is an in vitro or a mammalian cell RNase H cleavage assay.

9. The nucleic acid duplex of claim 1, wherein DNA of said RNA:DNA duplex of said second region consists of 4-40 deoxyribonucleotides that base pair with ribonucleotides, and wherein said nick is positioned on one of said first oligonucleotide strand or said second oligonucleotide strand between immediately adjacent deoxyribonucleotides.

10. The nucleic acid duplex of claim 1, wherein DNA of said RNA:DNA duplex of said second region consists of 4-40 deoxyribonucleotides that base pair with ribonucleotides, and wherein said nick is positioned on one of said first oligonucleotide strand or said second oligonucleotide strand between a deoxyribonucleotide that is immediately adjacent to a ribonucleotide.

11. The isolated nucleic acid duplex of claim 9 or 10, wherein DNA said RNA:DNA duplex

12. The nucleic acid duplex of claim 1, wherein DNA of said RNA:DNA duplex of said second region consists of 4-20 deoxyribonucleotides that base pair with ribonucleotides, and wherein said nick is positioned on one of said first oligonucleotide strand or said second oligonucleotide strand between immediately adjacent deoxyribonucleotides.

13. The nucleic acid duplex of claim 1, wherein DNA of said RNA:DNA duplex of said second region consists of 4-20 deoxyribonucleotides that base pair with ribonucleotides, and wherein said nick is positioned on one of said first oligonucleotide strand or said second oligonucleotide strand between a deoxyribonucleotide that is immediately adjacent to a ribonucleotide.

14. The nucleic acid of claim 1, wherein said first region comprises a duplex of at least 25 nucleotides in length.

15. The nucleic acid of claim 1, wherein said third region comprises a duplex of at least 25 nucleotides in length.

16. The isolated nucleic acid duplex of claim 1, wherein said second oligonucleotide strand of said first region is sufficiently complementary to a first target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell.

17. The isolated nucleic acid duplex of claim 16, wherein said first target RNA is selected from the group consisting of K-RAS, HPRT1, VEGF, VEGFR, EGF, EGFR and an HCV target RNA sequence.

18. The isolated nucleic acid duplex of claim 1, wherein said second oligonucleotide strand of said third region is sufficiently complementary to a second target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell.

19. The isolated nucleic acid duplex of claim 1, wherein said first oligonucleotide strand of said third region is sufficiently complementary to a second target RNA along at least 19 nucleotides of said first oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell.

20. The isolated nucleic acid duplex of claim 18 or claim 19, wherein said second target RNA is selected from the group consisting of K-RAS, HPRT1, VEGF, VEGFR, EGF, EGFR and an HCV target RNA sequence.

21. The isolated nucleic acid duplex of claim 20, wherein pairs of first and second target RNAs are selected from the group consisting of HPRT1 and K-RAS; VEGF and VEGFR; and EGF and EGFR.

22. The isolated nucleic acid duplex of claim 16, wherein said nucleic acid duplex reduces target gene expression in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.

23. The isolated nucleic acid duplex of claim 18 or claim 19, wherein said nucleic acid duplex reduces expression of said first target gene and said second target gene in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.

24. The isolated nucleic acid duplex of claim 1, wherein said second oligonucleotide strand of said first region possesses a 3' overhang of 1-4 nucleotides in length.

25. The isolated nucleic acid duplex of claim 1, wherein said first oligonucleotide strand of said third region possesses a 3' overhang of 1-4 nucleotides in length.

26. The isolated nucleic acid duplex of claim 24 or claim 25, wherein said 3' overhang is 1-3 nucleotides in length.

27. The isolated nucleic acid duplex of claim 26, wherein said 3' overhang is 1-2 nucleotides in length.

28. The isolated nucleic acid duplex of claim 24 or claim 25, wherein said nucleotides of said 3' overhang comprise a modified nucleotide.

29. The isolated nucleic acid duplex of claim 28, wherein said modified nucleotide residue is selected from the group consisting of 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O -[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH.sub.2--O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino and 2'-O-(N-methlycarbamate).

30. The isolated nucleic acid duplex of claim 28, wherein said modified nucleotide of said 3' overhang is a 2'-O-methyl ribonucleotide.

31. The isolated nucleic acid duplex of claim 28, wherein all nucleotides of said 3' overhang are modified nucleotides.

32. The isolated nucleic acid duplex of claim 28, wherein said 3' overhang is two nucleotides in length and wherein said modified nucleotide of said 3' overhang is a 2'-O-methyl modified ribonucleotide.

33. The isolated nucleic acid duplex of claim 1, wherein said second oligonucleotide strand of said first region, starting from the nucleotide residue of said second oligonucleotide strand of said first region that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand of said first region, comprises unmodified nucleotide residues at all positions from position 20 to the most 5' residue of said second oligonucleotide strand of said first region.

34. The isolated nucleic acid duplex of claim 1, wherein said first oligonucleotide strand of said third region, starting from the nucleotide residue of said first oligonucleotide strand of said third region that is complementary to the 5' terminal nucleotide residue of said second oligonucleotide strand of said third region, comprises unmodified nucleotide residues at all positions from position 20 to the most 5' residue of said first oligonucleotide strand of said third region.

35. The isolated nucleic acid duplex of claim 1, wherein the second oligonucleotide strand of said first region comprises modified nucleotides at positions 1, 2, and 3 from the 3' terminus of said second oligonucleotide strand of said first region.

36. The isolated nucleic acid duplex of claim 1, wherein the first oligonucleotide strand of said third region comprises modified nucleotides at positions 1, 2, and 3 from the 3' terminus of said first oligonucleotide strand of said third region.

37. The isolated nucleic acid duplex of claim 1, wherein at least the two most 3' nucleotide residues of said first oligonucleotide strand of said first region are deoxyribonucleotides that base pair with two deoxyribonucleotides of said second oligonucleotide strand of said first region.

38. The isolated nucleic acid duplex of claim 1, wherein at least the two most 5' nucleotide residues of said first oligonucleotide strand of said third region are deoxyribonucleotides that base pair with two deoxyribonucleotides of said second oligonucleotide strand of said third region.

39. The isolated nucleic acid duplex of claim 1, wherein the two most 3' nucleotide residues of said second oligonucleotide strand of said second region are modified ribonucleotides.

40. The isolated nucleic acid duplex of claim 1, wherein the two most 5' nucleotide residues of said second oligonucleotide strand of said second region are modified ribonucleotides.

41. The isolated nucleic acid duplex of claim 1, wherein said second oligonucleotide strand of said third region, starting from the most 3' nucleotide residue of said second oligonucleotide strand of said third region, comprises alternating modified and unmodified nucleotide residues.

42. The isolated nucleic acid duplex of claim 3, wherein the two most 5' nucleotide residues of said first oligonucleotide strand of said second region are modified ribonucleotides.

43. The isolated nucleic acid duplex of claim 3, wherein the two most 3' nucleotide residues of said first oligonucleotide strand of said second region are modified ribonucleotides.

44. The isolated nucleic acid duplex of claim 3, wherein said first oligonucleotide strand of said third region, starting from the 3' terminus of said first oligonucleotide strand of said third region, comprises alternating modified and unmodified nucleotide residues.

45. The isolated nucleic acid duplex of claim 1, wherein the 3' terminus of said first oligonucleotide strand of said third region and the 5' terminus of said second oligonucleotide strand of said third region form a blunt end.

46. The isolated nucleic acid duplex of claim 1, wherein at least one of positions 1, 2 or 3 from the 3' terminus of said 3' terminus of said first oligonucleotide strand of said third region is a deoxyribonucleotide.

47. The isolated nucleic acid duplex of claim 1, wherein said deoxynucleotide residues of said second region that comprise said RNA:DNA duplex are unmodified deoxyribonucleotides.

48. The isolated nucleic acid duplex of claim 1, wherein at least 50% of all deoxyribonucleotides of said nucleic acid duplex are unmodified deoxyribonucleotides.

49. The isolated nucleic acid duplex of claim 1, wherein said first oligonucleotide strand of said third region is attached to said second oligonucleotide strand of said third region by a nucleotide sequence, wherein said nucleotide sequence attaches the most 3' nucleotide of said first oligonucleotide strand of said third region that base pairs with a nucleotide of said second oligonucleotide strand of said third region to said second oligonucleotide strand nucleotide of said third region that base pairs with said most 3' nucleotide of said first oligonucleotide strand of said third region.

50. The isolated nucleic acid duplex of claim 49, wherein said nucleotide sequence that attaches said first oligonucleotide strand of said third region and said second oligonucleotide strand of said third region comprises a tetraloop.

51. The isolated nucleic acid duplex of claim 49, wherein said nucleotide sequence that attaches said first oligonucleotide strand of said third region and said second oligonucleotide strand of said third region comprises a hairpin, a chemical linker or an extended loop.

52. The isolated nucleic acid duplex of claim 1, wherein one or both of the first and second oligonucleotide strands of any of said first, second or third regions comprises a 5' phosphate.

53. The isolated nucleic acid duplex of claim 1, wherein said nucleic acid duplex is cleaved endogenously in a mammalian cell by RNase H.

54. The isolated nucleic acid duplex of claim 53, wherein said endogenous RNase H cleavage generates a nucleic acid duplex that is cleaved endogenously in said mammalian cell by Dicer.

55. The isolated nucleic acid of claim 53, wherein said endogenous RNase H cleavage generates two nucleic acid duplexes that are each cleaved endogenously in said mammalian cell by Dicer.

56. The isolated nucleic acid duplex of claim 1, wherein said nucleic acid duplex is cleaved endogenously in a mammalian cell by Dicer.

57. The isolated nucleic acid duplex of claim 1, wherein said nucleic acid duplex is cleaved endogenously in a mammalian cell to produce a double-stranded nucleic acid of 19-23 nucleotides in length that reduces target gene expression.

58. The isolated nucleic acid duplex of claim 1, wherein a nucleotide of said second or first oligonucleotide strand of any of said first, second or third regions is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

59. The isolated nucleic acid duplex of claim 1 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

60. The isolated nucleic acid duplex of claim 1, wherein at least 50% of said ribonucleotide residues of positions 1 to 23 of said first oligonucleotide strand that base pair with ribonucleotides of said second oligonucleotide strand to form a duplex are unmodified ribonucleotides.

61. The isolated nucleic acid duplex of claim 1, wherein at least 50% of all ribonucleotides of said nucleic acid duplex are unmodified ribonucleotides.

62. The isolated nucleic acid duplex of claim 1, wherein the first and second oligonucleotide strands of said third region are joined by a chemical linker.

63. The isolated nucleic acid duplex of claim 62, wherein said 3' terminus of said first oligonucleotide strand of said third region and said 5' terminus of said second oligonucleotide strand of said third region are joined by a chemical linker.

64. The isolated nucleic acid duplex of claim 1, wherein positions 24 and greater of said first oligonucleotide strand of said first region comprise between one and 12 deoxyribonucleotide residues, wherein each of said deoxynucleotide residues of said first oligonucleotide strand of said first region base pairs with a deoxyribonucleotide of said second oligonucleotide strand of said first region to form a duplex.

65. The isolated nucleic acid duplex of claim 1, wherein the first oligonucleotide strand of said first or third regions has a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95% or 100% complementary to the second oligonucleotide strand nucleotide sequence of said respective first or third regions.

66. An isolated nucleic acid duplex comprising: a first region comprising first and second oligonucleotide strands comprising ribonucleotides, each strand having 5' and 3' termini, wherein said first region forms a duplex of 23 and 35 ribonucleotides in length; a second region comprising first and second oligonucleotide strands, said second region comprising a DNA:DNA duplex having a length of DNA of 2-40 base pairs; and a third region comprising first and second oligonucleotide strands comprising ribonucleotides, each strand having 5' and 3' termini, wherein said third region forms a duplex of 23 and 35 ribonucleotides in length.

67. The nucleic acid of claim 66, wherein said first region comprises a duplex of at least 25 nucleotides in length.

68. The nucleic acid of claim 66, wherein said first region comprises a duplex of between 26 and 35 nucleotides in length.

69. The nucleic acid of claim 66, wherein said first region comprises a duplex of between 26 and 30 nucleotides in length.

70. The nucleic acid of claim 66, wherein said third region comprises a duplex of at least 25 nucleotides in length.

71. The nucleic acid of claim 66, wherein said third region comprises a duplex of between 26 and 35 nucleotides in length.

72. The nucleic acid of claim 66, wherein said third region comprises a duplex of between 26 and 30 nucleotides in length.

73. The isolated nucleic acid duplex of claim 66, wherein said second oligonucleotide strand of said first region is sufficiently complementary to a first target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell.

74. The isolated nucleic acid duplex of claim 73, wherein said first target RNA is selected from the group consisting of K-RAS, HPRT1, VEGF, VEGFR, EGF, EGFR and an HCV target RNA sequence.

75. The isolated nucleic acid duplex of claim 66, wherein said second oligonucleotide strand of said third region is sufficiently complementary to a second target RNA along at least 19 nucleotides of either said first or said second oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell.

76. The isolated nucleic acid duplex of claim 73, wherein said first oligonucleotide strand of said third region is sufficiently complementary to a second target RNA along at least 19 nucleotides of either said first or said second oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell.

77. The isolated nucleic acid duplex of claim 75 or claim 76, wherein said second target RNA is selected from the group consisting of K-RAS, HPRT1, VEGF, VEGFR, EGF, EGFR and an HCV target RNA sequence.

78. The isolated nucleic acid duplex of claim 77, wherein pairs of first and second target RNAs are selected from the group consisting of HPRT1 and K-RAS; VEGF and VEGFR; and EGF and EGFR.

79. The isolated nucleic acid duplex of claim 66, wherein said nucleic acid duplex reduces target gene expression in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.

80. The isolated nucleic acid duplex of claim 75 or claim 76, wherein said nucleic acid duplex reduces expression of said first target gene and said second target gene in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.

81. The isolated nucleic acid duplex of claim 66, wherein said second oligonucleotide strand of said first region possesses a 3' overhang of 1-4 nucleotides in length.

82. The isolated nucleic acid duplex of claim 66, wherein said first oligonucleotide strand of said third region possesses a 3' overhang of 1-4 nucleotides in length.

83. The isolated nucleic acid duplex of claim 81 or claim 82, wherein said 3' overhang is 1-3 nucleotides in length.

84. The isolated nucleic acid duplex of claim 83, wherein said 3' overhang is 1-2 nucleotides in length.

85. The isolated nucleic acid duplex of claim 81 or claim 82, wherein said nucleotides of said 3' overhang comprise a modified nucleotide.

86. The isolated nucleic acid duplex of claim 85, wherein said modified nucleotide residue is selected from the group consisting of 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O -[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH.sub.2--O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino and 2'-O-(N-methlycarbamate).

87. The isolated nucleic acid duplex of claim 85, wherein said modified nucleotide of said 3' overhang is a 2'-O-methyl ribonucleotide.

88. The isolated nucleic acid duplex of claim 85, wherein all nucleotides of said 3' overhang are modified nucleotides.

89. The isolated nucleic acid duplex of claim 85, wherein said 3' overhang is two nucleotides in length and wherein said modified nucleotide of said 3' overhang is a 2'-O-methyl modified ribonucleotide.

90. The isolated nucleic acid duplex of claim 66, wherein said second oligonucleotide strand of said first region, starting from the nucleotide residue of said second oligonucleotide strand of said first region that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand of said first region, comprises unmodified nucleotide residues at all positions from position 20 to the most 5' residue of said second oligonucleotide strand of said first region.

91. The isolated nucleic acid duplex of claim 66, wherein said first oligonucleotide strand of said third region, starting from the nucleotide residue of said first oligonucleotide strand of said third region that is complementary to the 5' terminal nucleotide residue of said second oligonucleotide strand of said third region, comprises unmodified nucleotide residues at all positions from position 20 to the most 5' residue of said first oligonucleotide strand of said third region.

92. The isolated nucleic acid duplex of claim 66, wherein the second oligonucleotide strand of said first region comprises modified nucleotides at positions 1, 2, and 3 from the 3' terminus of said second oligonucleotide strand of said first region.

93. The isolated nucleic acid duplex of claim 66, wherein the first oligonucleotide strand of said third region comprises modified nucleotides at positions 1, 2, and 3 from the 3' terminus of said first oligonucleotide strand of said third region.

94. The isolated nucleic acid duplex of claim 66, wherein at least the two most 3' nucleotide residues of said first oligonucleotide strand of said first region are deoxyribonucleotides that base pair with two deoxyribonucleotides of said second oligonucleotide strand of said first region.

95. The isolated nucleic acid duplex of claim 66, wherein at least the two most 5' nucleotide residues of said first oligonucleotide strand of said third region are deoxyribonucleotides that base pair with two deoxyribonucleotides of said second oligonucleotide strand of said third region.

96. The isolated nucleic acid duplex of claim 66, wherein the two most 3' nucleotide residues of said second oligonucleotide strand of said second region are modified ribonucleotides.

97. The isolated nucleic acid duplex of claim 66, wherein the two most 5' nucleotide residues of said second oligonucleotide strand of said second region are modified ribonucleotides.

98. The isolated nucleic acid duplex of claim 66, wherein said second oligonucleotide strand of said third region, starting from the most 3' nucleotide residue of said second oligonucleotide strand of said third region, comprises alternating modified and unmodified nucleotide residues.

99. The isolated nucleic acid duplex of claim 66, wherein the 3' terminus of said first oligonucleotide strand of said third region and the 5' terminus of said second oligonucleotide strand of said third region form a blunt end.

100. The isolated nucleic acid duplex of claim 66, wherein at least one of positions 1, 2 or 3 from the 3' terminus of said 3' terminus of said first oligonucleotide strand of said third region is a deoxyribonucleotide.

101. The isolated nucleic acid duplex of claim 66, wherein said deoxynucleotide residues of said second region that comprise said DNA:DNA duplex are unmodified deoxyribonucleotides.

102. The isolated nucleic acid duplex of claim 66, wherein at least 50% of all deoxyribonucleotides of said nucleic acid duplex are unmodified deoxyribonucleotides.

103. The isolated nucleic acid duplex of claim 66, wherein said first oligonucleotide strand of said third region is attached to said second oligonucleotide strand of said third region by a nucleotide sequence, wherein said nucleotide sequence attaches the most 3' nucleotide of said first oligonucleotide strand of said third region that base pairs with a nucleotide of said second oligonucleotide strand of said third region to said second oligonucleotide strand nucleotide of said third region that base pairs with said most 3' nucleotide of said first oligonucleotide strand of said third region.

104. The isolated nucleic acid duplex of claim 103, wherein said nucleotide sequence that attaches said first oligonucleotide strand of said third region and said second oligonucleotide strand of said third region comprises a tetraloop.

105. The isolated nucleic acid duplex of claim 103, wherein said nucleotide sequence that attaches said first oligonucleotide strand of said third region and said second oligonucleotide strand of said third region comprises a hairpin, a chemical linker or an extended loop.

106. The isolated nucleic acid duplex of claim 66, wherein one or both of the first and second oligonucleotide strands of any of said first, second or third regions comprises a 5' phosphate.

107. The isolated nucleic acid duplex of claim 66, wherein said nucleic acid duplex is cleaved endogenously in a mammalian cell by Dicer.

108. The isolated nucleic acid duplex of claim 107, wherein said nucleic acid duplex is cleaved twice endogenously in a mammalian cell by Dicer.

109. The isolated nucleic acid duplex of claim 66, wherein said nucleic acid duplex is cleaved endogenously in a mammalian cell to produce a double-stranded nucleic acid of 19-23 nucleotides in length that reduces target gene expression.

110. The isolated nucleic acid duplex of claim 66, wherein a nucleotide of said second or first oligonucleotide strand of any of said first, second or third regions is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

111. The isolated nucleic acid duplex of claim 66 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

112. The isolated nucleic acid duplex of claim 66, wherein at least 50% of said ribonucleotide residues of positions 1 to 23 of said first oligonucleotide strand that base pair with ribonucleotides of said second oligonucleotide strand to form a duplex are unmodified ribonucleotides.

113. The isolated nucleic acid duplex of claim 66, wherein at least 50% of all ribonucleotides of said nucleic acid duplex are unmodified ribonucleotides.

114. The isolated nucleic acid duplex of claim 66, wherein the first and second oligonucleotide strands of said third region are joined by a chemical linker.

115. The isolated nucleic acid duplex of claim 114, wherein said 3' terminus of said first oligonucleotide strand of said third region and said 5' terminus of said second oligonucleotide strand of said third region are joined by a chemical linker.

116. The isolated nucleic acid duplex of claim 66, wherein positions 24 and greater of said first oligonucleotide strand of said first region comprise between one and 12 deoxyribonucleotide residues, wherein each of said deoxynucleotide residues of said first oligonucleotide strand of said first region base pairs with a deoxyribonucleotide of said second oligonucleotide strand of said first region to form a duplex.

117. The isolated nucleic acid duplex of claim 66, wherein the first oligonucleotide strand of said first or third regions has a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95% or 100% complementary to the second oligonucleotide strand nucleotide sequence of said respective first or third regions.

118. The isolated nucleic acid duplex of any of claims 1 or 66, wherein said isolated nucleic acid duplex is at least 50% more effective at target gene inhibition in a mammalian cell contacted with a fixed concentration of said nucleic acid duplex than a corresponding bifunctional siRNA agent at the same concentration.

119. The isolated nucleic acid duplex of any of claims 1 or 66, wherein said isolated nucleic acid duplex possesses a duration of target gene inhibition in a mammalian cell contacted with a fixed concentration of said nucleic acid duplex that is at least 25% longer than a corresponding bifunctional siRNA agent at the same concentration.

120. A method for reducing expression of a first target gene and a second target gene in a cell, comprising: contacting a cell with an isolated nucleic acid duplex as claimed in any one of claims 1 or 66 in an amount effective to reduce expression of a first target gene and a second target gene in a cell more than two unattached reference dsRNAs.

121. A method for reducing expression of a first target gene and a second target gene in an animal, comprising: administering to an animal an isolated nucleic acid duplex as claimed in any one of claims 1 or 66 in an amount effective to reduce expression of a first target gene and a second target gene in a cell of the animal more than two unattached reference dsRNAs.

122. A pharmaceutical composition for reducing expression of a first target gene and a second target gene in a cell of a subject comprising an isolated nucleic acid duplex as claimed in any one of claims 1 or 66 in an amount effective to reduce expression of a first target gene and a second target gene in a cell, and a pharmaceutically acceptable carrier.

123. A pharmaceutical composition for reducing expression of a first target gene and a second target gene in a cell of a subject comprising an isolated nucleic acid duplex as claimed in any one of claims 1 or 66 in an amount effective to reduce expression of a first target gene and a second target gene in a cell more than two unattached reference dsRNAs, and a pharmaceutically acceptable carrier.

124. An isolated nucleic acid duplex comprising: a first region comprising first and second oligonucleotide strands comprising ribonucleotides, said first strand having a 5' terminus and said second strand having a 3' terminus, wherein the nucleotides of said first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; and a second region comprising a first oligonucleotide strand and a second oligonucleotide strand comprising a RNA:DNA duplex having a length of DNA sufficient to activate a detectable amount of RNase H cleavage of said second region in an RNase H cleavage assay, wherein said second region is covalently attached to said first region; wherein the 5' terminal residue of said second oligonucleotide strand or the 3' terminal residue of said first oligonucleotide strand of said second region is covalently attached to a functional group.

125. An isolated nucleic acid duplex comprising: a first region comprising a first oligonucleotide strand comprising ribonucleotides and having a 5' terminus and a second oligonucleotide strand comprising ribonucleotides and having a 3' terminus, wherein the nucleotides of said first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; and a second region comprising a RNA:DNA duplex, wherein said RNA:DNA duplex comprises a first oligonucleotide strand having a 3' terminus, wherein the most 5' nucleotide of said first oligonucleotide strand of said second region is covalently attached to the most 3' nucleotide of said first oligonucleotide strand of said first region, and a second oligonucleotide strand having a 5' terminus, wherein the most 3' nucleotide of said second oligonucleotide strand of said second region is covalently attached to the most 5' nucleotide of said second oligonucleotide strand of said first region, wherein said first oligonucleotide strand of said second region comprises between four and twenty deoxyribonucleotides that form a RNA:DNA duplex with ribonucleotides of said second oligonucleotide strand of said second region, wherein the 5' terminal residue of said second oligonucleotide strand of said second region is covalently attached to a functional group.

126. An isolated nucleic acid duplex comprising: a first region comprising a first oligonucleotide strand comprising ribonucleotides and having a 5' terminus and a second oligonucleotide strand comprising ribonucleotides and having a 3' terminus, wherein the nucleotides of said first and second oligonucleotide strands form a duplex of between 23 and 30 nucleotides in length; and a second region comprising a RNA:DNA duplex, wherein said RNA:DNA duplex comprises a first oligonucleotide strand having a 3' terminus, wherein the most 5' nucleotide of said first oligonucleotide strand of said second region is covalently attached to the most 3' nucleotide of said first oligonucleotide strand of said first region, and a second oligonucleotide strand having a 5' terminus, wherein the most 3' nucleotide of said second oligonucleotide strand of said second region is covalently attached to the most 5' nucleotide of said second oligonucleotide strand of said first region, wherein said second oligonucleotide strand of said second region comprises between four and twenty deoxyribonucleotides that form a RNA:DNA duplex with ribonucleotides of said first oligonucleotide strand of said second region, wherein the 3' terminal residue of said first oligonucleotide strand of said second region is covalently attached to a functional group.

127. The isolated nucleic acid duplex of claims 124-126, wherein said second oligonucleotide strand of said first region is sufficiently complementary to a target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said nucleic acid duplex is introduced into a mammalian cell.

128. The isolated nucleic acid duplex of claims 124-126, wherein said functional group is selected from the group consisting of a ligand for a cellular receptor, a protein localization sequence, an antibody; a nucleic acid aptamer, a vitamin or other co-factor, a polymer, a phospholipid, cholesterol, a polyamine, an intercalator, a reporter molecule, a polyamine, a polyamide, polyethylene glycol, polyether, a group that enhances a pharmacodynamic property of a nucleic acid agent, a group that enhances a pharmacokinetic property of a nucleic acid agent and an active drug substance.

129. The isolated nucleic acid duplex of claim 128, wherein said functional group is attached to said second region by a linking moiety.

130. The isolated nucleic acid duplex of claim 129, wherein said linking moiety is selected from the group consisting of a chemical linker and an extended loop.

131. The isolated nucleic acid duplex of claims 124-126, wherein said functional group improves formulation, biodistribution, adsorption, metabolism, pharmacodynamics or cellular uptake of said nucleic acid duplex.

132. A method for reducing expression of a target gene in a cell, comprising: contacting a cell with an isolated nucleic acid duplex as claimed in any one of claims 1, 6, and 124 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA.

133. A method for reducing expression of a target gene in an animal, comprising: administering to an animal an isolated nucleic acid duplex as claimed in any one of claims 1, 6, and 124 in an amount effective to reduce expression of a target gene in a cell of the animal in comparison to a reference dsRNA.

134. A pharmaceutical composition for reducing expression of a target gene in a cell of a subject comprising an isolated nucleic acid duplex as claimed in any one of claims 1, 6, and 124 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA and a pharmaceutically acceptable carrier.

135. A method of synthesizing an isolated nucleic acid duplex as claimed in any one of claims 1, 6, and 124, comprising chemically or enzymatically synthesizing said nucleic acid duplex.

136. A kit comprising the isolated nucleic acid duplex of any one of claims 1, 6, and 124 and instructions for its use.