WO2003070917A2

WO2003070917A2 - Rna interference mediated inhibition of myc and myb genes or genes of their respective pathways

Info

Publication number: WO2003070917A2
Application number: PCT/US2003/005326
Authority: WO
Inventors: James Mcswiggen; Leonid Beigelman
Original assignee: Sirna Therapeutics, Inc.
Priority date: 2002-02-20
Filing date: 2003-02-20
Publication date: 2003-08-28
Also published as: EP1448590A4; EP1448590A2; WO2003070917A3; AU2003213203A8; AU2003213203A1

Abstract

The present invention concerns methods and reagents useful in modulating Myc and/or Myb gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against c-Myc, N-Myc, L-Myc, c-Myb, b-Myb, and v-Myb genes. The small nucleic acid molecules are useful in the treatment of cancer and other conditions, diseases and disorders.

Description

RNA INTERFERENCE MEDIATED INHIBITION OF MYC AND MYB GENE

EXPRESSION OR EXPRESSION OF GENES INVOLVED IN THE MYC AND

MYB PATHWAYS USING SHORT INTERFERING NUCLEIC ACID (siNA)

This application claims the benefit of Beigelman, U.S. Application Serial No. 60/418,655, filed October 15, 2002; of Beigelman, U.S. Application Serial No. 60/358,580, filed February 20, 2002; of Beigelman, U.S. Application Serial No. 60/363,124, filed March 11, 2002; of Beigelman, U.S. Application Serial No. 60/386,782 filed June 6, 2002; of Beigelman, U.S. Application Serial No. 60/406,784, filed August 29, 2002; of Beigelman, U.S. Application Serial No. 60/408,378, filed September 5, 2002; of Beigelman, U.S. Application Serial No. 60/409,293, filed September 9, 2002; and of Beigelman, U.S. Application Serial No. 60/440,129, filed January 15, 2003. These applications are hereby incorporated by reference herein in their entireties, including the drawings.

Field Of The Invention The present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases that respond to the modulation of Myc and/or Myb gene expression and/or activity. The present invention also concerns compounds, compositions, and methods relating to conditions and diseases that respond to the modulation of expression and/or activity of genes involved in the Myc and/or Myb pathway. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against Myc genes, other genes involved in the Myc pathway, Myb genes, and other genes involved in the Myb pathway. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against c-Myc, N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, and v-Myb genes. Background Of The Invention

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

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

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al, 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir et al, 2001, Genes Dev., 15, 188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al, 2001, Science, 293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single- stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al, 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al, 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol, 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al, 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al, 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21 -nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al, 2001, EMBO J., 20, 6877) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 -nucleotide siRNA duplexes are most active when containing 3'-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2'-deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3'-terminal siRNA overhang nucleotides with 2'-deoxy nucleotides (2'-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity, hi addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5'-end of the siRNA guide sequence rather than the 3'-end of the guide sequence (Elbashir et al, 2001, EMBO J., 20, 6877). Other studies have indicated that a 5 '-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5'-phosphate moiety on the siRNA (Nykanen et al, 2001, Cell, 107, 309).

Studies have shown that replacing the 3 '-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two nucleotide 3 '-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well- tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al, 2001, EMBO J., 20, 6877). hi addition, Elbashir et al, supra, also report that substitution of siRNA with 2'-O-methyl nucleotides completely abolishes RNAi activity. Li et al, International PCT Publication No. WO 00/44914, and Beach et al, Mtemational PUT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA. Kreutzer et al, Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2'-amino or 2'-O-methyl nucleotides, and nucleotides containing a 2'-O or 4'-C methylene bridge. However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in siRNA molecules.

Parrish et al, 2000, Molecular Cell, 6, 1977-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi. Further, Parrish et al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081. The authors also tested certain modifications at the 2'-position of the nucleotide sugar in the long siRNA transcripts and found that substituting deoxynucleotides for ribonucleotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil substitution appeared to be tolerated, Parrish reported that inosine produced a substantial decrease in interference activity when incoφorated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al, International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression usmg endogenously-derived dsRNA. Tuschl et al, International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger of activating interferon response. Li et al, International PCT Publication No. WO 00/44914, describe the use of specific dsRNAs for attenuating the expression of certain target genes. Zernicka-Goetz et al, International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain dsRNA molecules. Fire et al, International PCT Publication No. WO 99/32619, describe particular methods for introducing certain dsRNA molecules into cells for use in inhibiting gene expression. Plaetinck et al, International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al, International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Deschamps Depaillette et al, hitemational PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al, hitemational PCT Publication No. 99/53050, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et al, hitemational PCT Publication No. WO 01/49844, describe specific DNA constructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al, 2000, Molecular Cell, 6, 1977-1087, describe specific chemically-modified siRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, mtemational PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al, hitemational PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al, hitemational PCT Publication No. WO 01/53475, describe certain methods for isolating a Neurospora silencing gene and uses thereof. Reed et al, hitemational PCT Publication No. WO 01/68836, describe certain methods for gene silencing in plants. Honer et al, International PCT Publication No. WO 01/70944, describe certain methods of drag screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al, International PCT Publication No. WO 01/72774, describe certain Drosophila-denved gene products that may be related to RNAi in Drosophila. Arndt et al, hitemational PCT Publication No. WO 01/92513, describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al, International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constracts. Pachuk et al, hitemational PCT Publication No. WO 00/63364, and Satishchandran et al, hitemational PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain dsRNAs. Echeverri et al, hitemational PCT Publication No. WO 02/38805, describe certain C. elegans genes identified via RNAi. Kreutzer et al, International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 Bl describes certain methods for inhibiting gene expression using RNAi. Graham et al, hitemational PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al, US 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain siRNA constructs that mediate RNAi.

SUMMARY OF THE INVENTION This invention relates to compounds, compositions, and methods useful for modulating the expression of genes associated with mitogen Myc gene expression pathways by RNA interference (RNAi) using small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short haiφin RNA (shRNA) molecules. The invention also relates to compounds, compositions, and methods useful for modulating the expression of genes associated with Myb gene expression pathways by RNA interference (RNAi) using short interfering nucleic acid (siNA) molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short haiφin RNA (shRNA) molecules and methods used to modulate the expression of c-Myc, N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, v-Myb, and genes involved in the Myc pathway such as cyclin DI and D2, cyclin E, CDK4, ana caczo v, <_^ Z and UDK4 genes. A siNA of the invention can be unmodified or chemically-modified. A siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating Myc and/or Myb gene expression or activity in cells by RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA having multiple chemical modifications retains its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

hi one embodiment, the invention features about one or more siNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding Myc proteins, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as Myc. In another embodiment, the invention features about one or more siNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding Myb proteins, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table II, referred to herein generally as Myb. The description below of the various aspects and embodiments of the invention is provided with reference to exemplary Myc and Myb genes. However, the various aspects and embodiments are also directed to other Myc and Myb genes referred to by Accession number in Tables I and II respectively. The various aspects and embodiments are also directed to other genes that are involved in the Myc and Myb pathways of gene expression. Those additional genes can be analyzed for target sites using the methods described for Myc and Myb genes herein. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein. Chemical modifications as shown in Table IX or otherwise described herein can be applied to any siNA construct of the invention. In one embodiment, the invention features a siNA molecule that down-regulates expression of a Myc gene, for example, wherein the Myc gene comprises Myc encoding sequence.

h another embodiment, the invention features a siNA molecule that down-regulates expression of a Myb gene, for example, wherein the Myb gene comprises Myb encoding sequence.

h one embodiment, the invention features a siNA molecule having RNAi activity against Myc RNA, wherein the siNA molecule comprises a sequence complementary to any RNA having Myc encoding sequence, such as those sequences having GenBank Accession Nos. shown in Table I. Chemical modifications as shown in Table IX or otherwise described herein can be applied to any siNA construct of the invention.

In another embodiment, the invention features a siNA molecule having RNAi activity against a Myc gene, wherein the siNA molecule comprises nucleotide sequence complementary to nucleotide sequence of a Myc gene, such as those Myc sequences having GenBank Accession Nos. shown in Table I. h another embodiment, a siNA molecule of the invention includes nucleotide sequence that can interact with nucleotide sequence of a Myc gene and thereby mediate silencing of Myc gene expression, for example wherein the siNA mediates regulation of Myc gene expression by cellular processes that modulate the chromatin stracture of the Myc gene and prevent transcription of the Myc gene. Chemical modifications as shown in Table IX or otherwise described herein can be applied to any siNA constract of the invention.

hi another embodiment, the invention features a siNA molecule comprising nucleotide sequence, for example nucleotide sequence in the antisense region of the siNA molecule, that is complementary to a nucleotide sequence or portion of sequence of a Myc gene. In another embodiment, the invention features a siNA molecule comprising a region, for example the antisense region of the siNA construct, complementary to a sequence or portion of sequence comprising a Myc gene sequence.

In another embodiment, the invention features a siNA molecule having RNAi activity against Myb RNA, wherein the siNA molecule comprises a sequence complementary to any RNA having Myb encoding sequence, such as those sequences navmg ureiutsanK Accession INOS. snown m laDie 11. Chemical modifications as shown in Table IX or otherwise described herein can be applied to any siNA construct of the invention.

hi another embodiment, the invention features a siNA molecule having RNAi activity against a Myb gene, wherein the siNA molecule comprises nucleotide sequence complementary to nucleotide sequence of a Myb gene, such as those Myb sequences having GenBank Accession Nos. shown in Table II. hi another embodiment, a siNA molecule of the invention includes nucleotide sequence that can interact with nucleotide sequence of a Myb gene and thereby mediate silencing of Myb gene expression, for example wherein the siNA mediates regulation of Myb gene expression by cellular processes that modulate the chromatin stracture of the Myb gene and prevent transcription of the Myb gene. Chemical modifications as shown in Table IX or otherwise described herein can be applied to any siNA construct of the invention.

In another embodiment, the invention features a siNA molecule comprising nucleotide sequence, for example nucleotide sequence in the antisense region of the siNA molecule, that is complementary to a nucleotide sequence or portion of sequence of a Myb gene, hi another embodiment, the invention features a siNA molecule comprising a region, for example the antisense region of the siNA construct, complementary to a sequence or portion of sequence comprising a Myb gene sequence.

In one embodiment, the antisense region of Myc siNA constracts can comprise a sequence complementary to sequence having any of SEQ ID NOs. 1-118 or 595-598. The antisense region can also comprise sequence having any of SEQ ID NOs. 119-236, 610-613, 618-621, 626-629, 668, 670, 672, 674, 676, or 677. In another embodiment, the sense region of Myc constructs can comprise sequence having any of SEQ ID NOs. 1- 118, 595-598, 606-609, 614-611, 622-625, 667, 669, 671, 673, or 675. The sense region can comprise a sequence of SEQ ID NO. 656 and the antisense region can comprise a sequence of SEQ ID NO. 657. The sense region can comprise a sequence of SEQ ID NO. 658 and the antisense region can comprise a sequence of SEQ JJD NO. 659. The sense region can comprise a sequence of SEQ ID NO. 660 and the antisense region can comprise a sequence of SEQ ID NO. 661. The sense region can comprise a sequence of SEQ ID NO. 662 and the antisense region can comprise a sequence of SEQ ID NO. 663. ine sense region can compnse a sequence of SEQ ID NO. 664 and the antisense region can comprise a sequence of SEQ ID NO. 665. The sense region can comprise a sequence of SEQ ID NO. 662 and the antisense region can comprise a sequence of SEQ ID NO. 666.

h one embodiment, the antisense region of Myb siNA constracts can comprise a sequence complementary to sequence having any of SEQ ID NOs. 237-415, 599-602, or 605. The antisense region can also comprise sequence having any of SEQ ID NOs. 416- 594, 603, 604, 634, 635, 640-643, 648-651, 679, 681, 683, 685, 687, or 688. In another embodiment, the sense region of Myb constracts can comprise sequence having any of SEQ ID NOs. 237-415, 599-602, 630-633, 636-639, 644-647, 605, 678, 680, 682, 684, or 686. The sense region can comprise a sequence of SEQ ID NO. 656 and the antisense region can comprise a sequence of SEQ ID NO. 657. The sense region can comprise a sequence of SEQ ID NO. 658 and the antisense region can comprise a sequence of SEQ ID NO. 659. The sense region can comprise a sequence of SEQ ID NO. 660 and the antisense region can comprise a sequence of SEQ ID NO. 661. The sense region can comprise a sequence of SEQ ID NO. 662 and the antisense region can comprise a sequence of SEQ ID NO. 663. The sense region can comprise a sequence of SEQ ID NO. 664 and the antisense region can comprise a sequence of SEQ ID NO. 665. The sense region can comprise a sequence of SEQ ID NO. 662 and the antisense region can comprise a sequence of SEQ ID NO. 666.

h one embodiment, a siNA construct of the invention can comprise any of SEQ ID

NOs. 1-688. The sequences shown in SEQ ID NOs: 1-688 are not limiting. A siNA molecule of the invention can comprise any contiguous Myc and/or Myb sequence (e.g., about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) contiguous Myc and/or Myb nucleotides).

In one embodiment, the invention features a siNA molecule comprising a sequence, for example the antisense sequence of the siNA constract, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I. Chemical modifications as shown in Table IX or otherwise described herein can be applied to any siNA constract of the invention.In another embodiment, the invention features a siNA molecule comprising a sequence, for example the antisense sequence oi me SU A constract, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table II. Chemical modifications as shown in Table IX or otherwise described herein can be applied to any siNA construct of the invention.

In one embodiment of the invention a siNA molecule comprises an antisense strand having about 19 to about 29 nucleotides, wherein the antisense strand is complementary to a RNA sequence encoding a Myc protein, and wherein said siNA further comprises a sense strand having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences with at least about 19 complementary nucleotides.

In another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, wherein the antisense region is complementary to a RNA sequence encoding a Myc protein, and wherein said siNA further comprises a sense region having about 19 to about 29 nucleotides, wherein said sense region and said antisense region comprise a linear molecule with at least about 19 complementary nucleotides.

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

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

In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a Myc gene. Because related genes typically share some degree of sequence homology with each other, siNA molecules can be designed to target a class of Myc genes or alternately specific Myc genes by selecting sequences mat are eitner snared amongst different Myc targets or alternatively that are unique for a specific Myc target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of Myc RNA sequence having homology between several Myc genes so as to target several Myc genes (e.g., splice variants, mutant genes etc.) with one siNA molecule. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific Myc RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment of the invention a siNA molecule comprises an antisense strand having about 19 to about 29 nucleotides, wherein the antisense strand is complementary to a RNA sequence encoding a Myb protein, and wherein said siNA further comprises a sense strand having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences with at least about 19 complementary nucleotides.

hi another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, wherein the antisense region is complementary to a RNA sequence encoding a Myb protein, and wherein said siNA further comprises a sense region having about 19 to about 29 nucleotides, wherein said sense region and said antisense region comprise a linear molecule with at least about 19 complementary nucleotides.

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

In another embodiment, a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a Myb protein. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a Myb gene or a portion thereof. h one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a Myb gene. Because related genes typically share some degree of sequence homology with each other, siNA molecules can be designed to target a class of Myb genes or alternately specific Myb genes by selecting sequences that are either shared amongst different Myb targets or alternatively that are unique for a specific Myb target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of Myb RNA sequence having homology between several Myb genes so as to target several Myb genes (e.g., splice variants, mutant genes etc.) with one siNA molecule, h another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific Myb RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a Myc gene. Because the c-Myc, N-Myc and L-Myc genes as a group typically share some degree of homology with each other, siNA molecules can be designed to target a class of Myc genes or alternately specific Myc genes by selecting sequences that are either shared amongst different Myc targets or that are alternately unique for a specific Myc target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of Myc RNA sequence having homology between several Myc genes so as to target several Myc targets with one siNA molecule. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific Myc RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a Myb gene. Because the c-Myb, a-Myb, b- Myb, and v-Myb genes as a group typically share some degree of sequence homology with each other, siNA molecules can be designed to target a class of Myb genes or alternately specific Myb genes by selecting sequences that are either shared amongst different Myb targets or that are alternately unique for a specific Myb target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of Myb RNA sequence having homology between several Myb genes so as to target several lviyo targets wi one SUN A molecule, in another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific Myb RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

hi one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplexes containing about 19 base pairs between oligonucleotides comprising about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24 or 25) nucleotides. In one embodiment, siNA molecules of the invention comprise duplexes with overhanging ends of about 1-3 (e.g., about 1, 2, or 3) nucleotides, for example about 21 -nucleotide duplexes with about 19 base pairs and 3'-terminal mononucleotide, dinucleotide, or trinucleotide overhangs.

In one embodiment, the invention features about one or more chemically-modified siNA constructs having specificity for Myc and/or Myb expressing nucleic acid molecules, such as RNA encoding a Myc and/or Myb protein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incoφoration. These chemical modifications, when used in various siNA constracts, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al, supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.

hi one embodiment, a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, and/or bioavailability. For example, a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, a siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percent modification can be based upon the total number of nucleotides present in the single stranded siNA molecules. Likewise, if the siNA molecule is double stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.

hi a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity in humans.

The antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3'-end of said antisense region. The antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5'-end of said antisense region. The 3'-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. The 3 '-terminal nucleotide overhangs can comprise about one or more universal base ribonucleotides. The 3 '-terminal nucleotide overhangs can comprise about one or more acyclic nucleotides. One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to a RNA or DNA sequence encoding Myc and/or Myb and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.

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

Myc and/or Myb inside a cell or reconstituted in vitro system, wherein the chemical modification comprises about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbone modified internucleotide linkage having Formula I:

wherein each RI and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y, and Z are optionally not all O.

The chemically-modified internucleotide linkages having Formula I, for example wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically- modified internucleotide linkages having Formula I at the 3 '-end, the 5'-end, or both of tne ό' ana 5 '-ends oi tne sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5'-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands, hi one non-limiting example, an exemplary siNA molecule of the invention can comprise about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands, hi another embodiment, a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non- nucleotide having any of Formulae I-VII.

h one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against Myc and/or Myb inside a cell or reconstituted in vitro system, wherein the chemical modification comprises about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, RI 1 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O- aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S=O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2- aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non- nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5 -nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise about one or more chemically-modified nucleotide or non-nucleotide of Formula II at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5 '-end of the sense strand, the antisense strand, or both strands, h anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3'-end of the sense strand, the antisense strand, or both strands.

h one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against Myc and/or Myb inside a cell or reconstituted in vitro system, wherein the chemical modification comprises about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III:

wherem each R3, R4, R5, R6, R7, R8, RIO, RI 1 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O- aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S=O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2- aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5- nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Fomiula III can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise about one or more chemically-modified nucleotide or non-nucleotide of

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

In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3'-3', 3'-2', 2'-3', or 5'-5' configuration, such as at the 3'-end, the 5'- end, or both of the 3' and 5'-ends of one or both siNA strands. In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against Myc and/or Myb inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a 5'-terminal phosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; wherein each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo; and wherein W, X, Y and Z are not all O.

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

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against Myc and/or Myb inside a cell or reconstituted in vitro system, wherein the chemical modification comprises about one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise about one or more phosphorothioate internucleotide linkages at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands. In one non-limiting example, an exemplary siNA molecule of the invention can comprise about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises about one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3,

4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'- ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense strand, hi another embodiment, about one or more, for example about 1, 2, 3, 4,

5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without about one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at me ύ -enα, tne rv-end, or ootn ol the 3'- and 5'-ends, being present in the same or different strand.

hi another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, or more) 2'- deoxy, 2'-0-methyl, 2 '-deoxy-2 '-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5'-end, or both of the 3'- and 5'-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense strand. In another embodiment, about one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'- fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends, being present in the same or different strand.

hi one embodiment, the invention features a siNA molecule, wherein the antisense strand comprises about one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fιuoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'- ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense strand, i another embodiment, about one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without about one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-ιTuoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 '-end, the 5 '-end, or both of the 3'- and 5 '-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense strand, hi another embodiment, about one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl and/or 2 '-deoxy-2 '-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3 '-end, the 5'-end, or both of the 3'- and 5'-ends, being present in the same or different strand.

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

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

In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand is about 18 to about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in length, wherein the duplex has about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the chemical modification comprises a stracture having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3 '-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs. In another embodiment, a siNA molecule of the invention comprises a single stranded haiφin stracture, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification comprising a stracture having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a haiφin stracture having about 19 base pairs and a 2-nucleotide 3 '-terminal nucleotide overhang. In another embodiment, a linear haiφin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear haiφin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3 '-terminal overhangs, such as 3 '-terminal nucleotide overhangs comprising about 2 nucleotides. hi another embodiment, a siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification, which comprises a stracture having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I — VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped stracture having about 19 base pairs and 2 loops.

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

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

wherein each R3, R4, R5, R6, R7, R8, R10, Rll, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S- alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl- OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O- aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S=O, CHF, or CF2.

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

wherein each R3, R4, R5, R6, R7, R8, RIO, Rl l, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S- alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl- OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O- aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S=O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siNA molecule of the invention.

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

wherein each n is independently an integer from 1 to 12, each RI, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl- OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl- O-alkyl, ON02, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O- aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having Formula I, and RI, R2 or R3 serves as points of attachment to the siNA molecule of the invention.

hi another embodiment, the invention features a compound having Formula VII, wherein RI and R2 are hydroxyl (OH) groups, n = 1, and R3 comprises O and is the point of attachment to the 3'-end, the 5'-end, or both of the 3' and 5'-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as "glyceryl" (for example modification 6 in Figure 10).

In another embodiment, a moiety having any of Formula V, VI or VII of the invention is at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of a siNA molecule of the invention. For example, a moiety having Formula V, VI or VII can be present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense strand, the sense strand, or both antisense and sense strands of the siNA molecule, hi addition, a moiety having Formula VII can be present at the 3'-end or the 5'-end of a haiφin siNA molecule as described herein.

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

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

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

h one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., about one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and where any (e.g., about one or more or all) purine nucleotides present in the sense region are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., about one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and where any (e.g., about one or more or all) purine nucleotides present in the sense region are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides), wherem any nucleotides comprising a 3 '-terminal nucleotide overhang that are present in said sense region are 2'-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., about one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately ' a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any (e.g., about one or more or all) purine nucleotides present in the antisense region are 2'-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl purine nucleotides).

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

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., about one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and where any (e.g., about one or more or all) purine nucleotides present in the antisense region are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'- deoxy purine nucleotides).

h one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against Myc and/or Myb inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where about one or more pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine liuuieuuues , anu wnere aoout one or more puπne nucleotides present m the sense region are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides), and inverted deoxy abasic modifications that are optionally present at the 3'- end, the 5 '-end, or both of the 3' and 5 '-ends of the sense region, the sense region optionally further comprising a 3'-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxyribonucleotides; and wherein the chemically-modified short interfering nucleic acid molecule comprises an antisense region, where about one or more pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein about one or more purine nucleotides present in the antisense region are 2'-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2'- O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3 '-end, the 5 '-end, or both of the 3' and 5'-ends of the antisense sequence, the antisense region optionally further comprising a 3'-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxynucleotides, wherein the overhang nucleotides can further comprise about one or more (e.g., about 1, 2, 3, or 4 ) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in Figures 4, 5, and 12 and Tables IV, VI, VII and VIII herein.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against Myc and/or Myb inside a cell or reconstituted in vitro system, wherein the siNA comprises a sense region, where about one or more pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and where about one or more purine nucleotides present in the sense region are purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides), and inverted deoxy abasic modifications that are optionally present at the 3'-end, the 5'-end, or both of the 3' and 5 '-ends of the sense region, the sense region optionally further comprising a 3'- terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2'- deoxyribonucleotides; and wherein the siNA comprises an antisense region, where about one or more pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy- 2'-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2'-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-0-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2'- O-methyl purine nucleotides), and a teπninal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3 '-end, the 5 '-end, or both of the 3' and 5'-ends of the antisense sequence, the antisense region optionally further comprising a 3'-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxynucleotides, wherein the overhang nucleotides can further comprise about one or more (e.g., about 1, 2, 3, or 4 ) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in Figures 4, 5, and 12 and Tables IV, VI, VII and VIII herein.In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against Myc and/or Myb inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where about one or more pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fiuoro pyrimidine nucleotides), and for example where about one or more purine nucleotides present in the sense region are selected from the group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'- methoxyethyl nucleotides, 4 '-thionucleotides, and 2'-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2 '-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2 '-methoxyethyl nucleotides, 4 '-thionucleotides, and 2 '-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2 '-methoxyethyl nucleotides, 4 '-thionucleotides, and 2 '-O-methyl nucleotides), and wherein inverted deoxy abasic modifications are optionally present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense region, the sense region optionally further comprising a 3'-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxyribonucleotides; and wherein the chemically-modified short interfering nucleic acid molecule comprises an antisense region, where about one or more pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein about one or more purine nucleotides present in the antisense region are selected from the group consisting of 2 '-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2 '-methoxyethyl nucleotides, 4 '-thionucleotides, and 2'- O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2 '-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'- methoxyethyl nucleotides, 4 '-thionucleotides, and 2 '-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2 '-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2 '-methoxyethyl nucleotides, 4'- thionucleotides, and 2 '-O-methyl nucleotides), and a temiinal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense sequence, the antisense region optionally further comprising a 3 '-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxynucleotides, wherein the overhang nucleotides can further comprise about one or more (e.g., about 1, 2, 3, or 4) phosphorothioate internucleotide linkages.

In another embodiment, any modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also in optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer- Verlag ed., 1984). As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also in optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Non- limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides); 2'- methoxyethoxy (MOE) nucleotides; 2 '-deoxy-2 '-fluoro nucleotides, 2 '-deoxy-2 '-chloro nucleotides, 2'-azido nucleotides, and 2'-O-methyl nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against Myc and/or Myb inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule, h another embodiment, the conjugate is covalently attached to the chemically- modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3 '-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, h one embodiment, the conjugate molecule is attached both the 3 '-end and 5 '-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a poly ethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al, U.S. Serial No. 10/201,394, incoφorated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constracts while at the same time maintaining the ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen siNA constracts that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.

hi one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non- nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of > 2 nucleotides in length, for example 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In one embodiment, the nucleotide linker can be a nucleic acid aptamer. By "aptamer" or "nucleic acid aptamer" as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al, 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol, 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol, 74, 27; Hermami and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.

In one embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.,polyethylene glycols such as those having about 2 to about 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 25:6353 and Nucleic Acids Res. 1987, 25:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 223:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 223:5109; Ma et al, Nucleic Acids Res. 1993, 22:2585 and Biochemistry 1993, 32:1151; Durand et al, Nucleic Acids Res. 1990, 25:6353; McCurdy et al, Nucleosides & Nucleotides 1991, 20:287; Jschke et al, Tetrahedron Lett. 1993, 54:301; Ono et al, Biochemistry 1991, 30:9914; Arnold et al, mtemational Publication No. WO 89/02439; Usman et al, hitemational Publication No. WO 95/06731; Dudycz et al, hitemational Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc 1991, 223:4000, all hereby incoφorated by reference herein. A "non-nucleotide" further means any group or compound that can be incoφorated into a nucleic acid chain in the place of about one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the Cl position of the sugar.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides. All positions within the siNA can include chemically modified nucleotides and/or non- nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence. In another embodiment, the single stranded siNA molecule of the invention comprises a 5 '-terminal phosphate group, hi another embodiment, the single stranded siNA molecule of the invention comprises a 5 '-terminal phosphate group and a 3 '-terminal phosphate group (e.g., a 2',3 '-cyclic phosphate), hi another embodiment, the single stranded siNA molecule of the invention comprises about 19 to about 29 nucleotides. h one embodiment, the single stranded siNA molecule of the invention comprises about one or more chemically-modified nucleotides or non- nucleotides described herein. For example, all the positions within the siNA molecule can include chemically-modified nucleotides such as nucleotides having any of Formulae I-VII, or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained. h one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein about one or more pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fiuoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2'-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl purine nucleotides), and a teπninal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3 '-end of the siNA molecule, wherein the terminal nucleotides can further comprise about one or more (e.g., about 1, 2, 3, or 4 ) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5 '-terminal phosphate group.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein about one or more pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3 '-end of the siNA molecule, wherein the terminal nucleotides can further comprise about one or more (e.g., about 1, 2, 3, or 4 ) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5 '-terminal phosphate group.

hi one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein about one or more pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides), and a terminal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3 '-end of the siNA molecule, wherein the terminal nucleotides can further comprise about one or more (e.g., about 1, 2, 3, or 4 ) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5 '-terminal phosphate group.

hi one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the siNA molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein about one or more pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2 '-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides are 2 '-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2 '-methoxyethyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense sequence, the siNA optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3'-end of the siNA molecule, wherein the terminal nucleotides can further comprise about one or more (e.g., about 1, 2, 3, or 4 ) phosphorothioate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5'-terminal phosphate group.

In another embodiment, any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer- Verlag ed., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.

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

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

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

hi one embodiment, the invention features a method of modulating the expression of a Myc and/or Myb gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the Myc and/or Myb gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the Myc and/or Myb gene in the tissue explant. hi another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the Myc and/or Myb gene in that organism.

hi one embodiment, the invention features a method of modulating the expression of a Myc and/or Myb gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the Myc and/or Myb gene and wherein the sense strand sequence of the siNA comprises a sequence identical to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the Myc and/or Myb gene in the tissue explant. h another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another orgamsm under conditions suitable to modulate the expression of the Myc and/or Myb gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one Myc and/or Myb gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the Myc and/or Myb genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the Myc and/or Myb genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the Myc and/or Myb genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a Myc and/or Myb gene in an organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the Myc and/or Myb gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the Myc and/or Myb gene in the organism.

In another embodiment, the invention features a method of modulating the expression of more than one Myc and/or Myb gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the Myc and/or Myb genes; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the Myc and/or Myb genes in the organism.

hi one embodiment, the invention features a method for modulating the expression of a Myc and/or Myb gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the Myc and/or Myb gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the Myc and/or Myb gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one Myc and/or Myb gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the Myc and/or Myb gene; and (b) contacting the siNA molecule with a cell in vitro or in vivo under conditions suitable to modulate the expression of the Myc and/or Myb genes in the cell.

In one embodiment, the invention features a method of modulating the expression of a Myc and/or Myb gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the Myc and/or Myb gene; and (b) contacting the siNA molecule with a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the Myc and/or Myb gene in the tissue explant. hi another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the Myc and/or Myb gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one Myc and/or Myb gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the Myc and/or Myb gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the Myc and/or Myb genes in the tissue explant. hi another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the Myc and/or Myb genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a Myc and/or Myb gene in an organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the Myc and/or Myb gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the Myc and/or Myb gene in the organism.

In another embodiment, the invention features a method of modulating the expression of more than one Myc and/or Myb gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the Myc and/or Myb gene; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the Myc and/or Myb genes in the organism.

In one embodiment, the invention features a method of modulating the expression of a Myc and/or Myb gene in an organism comprising contacting the organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the Myc and/or Myb gene in the organism.

In another embodiment, the invention features a method of modulating the expression of more than one Myc and/or Myb gene in an organism comprising contacting the organism with about one or more siNA molecules of the invention under conditions suitable to modulate the expression of the Myc and/or Myb genes in the organism.

The siNA molecules of the invention can be designed to inhibit target (Myc and/or Myb) gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymoφhism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as Myc and/or Myb genes. As such, siNA molecules targeting multiple Myc and/or Myb targets can provide increased therapeutic effect, hi addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, the progression and/or maintenance of cancer or other proliferative diseases and disorders.

In one embodiment, siNA molecule(s) and/or methods of the invention are used to inhibit the expression of gene(s) that encode RNA refeπed to by Genbank Accession, for example Myc and/or Myb genes encoding RNA sequence(s) refeπed to herein by Genbank Accession number, for example Genbank Accession Nos. shown in Table I and Table II. Chemical modifications as shown in Table IX or otherwise described herein can be applied to any siNA constract of the invention.

hi one embodiment, the invention features a method comprising: (a) generating a library of siNA constracts having a predetermined complexity; and (b) assaying the siNA constracts of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence, hi another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length, hi one embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, hi yet another embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. The fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. In another embodiment, the target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constracts having a predetermined complexity, such as of 4^N, where N represents the number of base paired nucleotides in each of the siNA constract strands (eg. for a siNA construct having about 21 nucleotide sense and antisense strands with about 19 base pairs, the complexity would be 4¹⁹); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target Myc and/or Myb RNA sequence, h another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In oneembodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, hi one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 7 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of Myc and/or Myb RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target Myc and/or Myb RNA sequence. The target Myc and/or Myb RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

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

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

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

In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting about one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with about one or more other therapeutic compounds. In one embodiment, the invention features a method for reducing or preventing tissue rejection in a subject comprising administering to the subject a composition of the invention under conditions suitable for the reduction or prevention of tissue rejection in the subject.

hi another embodiment, the invention features a method for validating a Myc and/or Myb gene target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of a Myc and/or Myb target gene; (b) introducing the siNA molecule into a cell, tissue, or organism under conditions suitable for modulating expression of the Myc and/or Myb target gene in the cell, tissue, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, or organism.

In another embodiment, the invention features a method for validating a Myc and/or Myb gene target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of a Myc and/or Myb target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the Myc and/or Myb target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.

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

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

h one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a Myc and/or Myb target gene in a cell, tissue, or organism, hi another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one Myc and/or Myb target gene in a cell, tissue, or organism.

In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a Myc and/or Myb target gene in a biological system. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one Myc and/or Myb target gene in a biological system.

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

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

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

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

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

hi another embodiment, the invention features a method for making a double- stranded siNA molecule in a single synthetic process, comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5 '-protecting group, for example a 5'-O-dimethoxytrityl group (5'-O-DMT), remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example, using a trityl-on synthesis strategy as described herein.

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

hi one embodiment, the invention features siNA constracts that mediate RNAi against Myc and/or Myb, wherein the siNA constract comprises about one or more chemical modifications, for example about one or more chemical modifications having any of Fonnulae I-VII or any combination thereof that increases the nuclease resistance of the siNA constract.

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

In one embodiment, the invention features siNA constructs that mediate RNAi against Myc and/or Myb, wherein the siNA constract comprises about one or more chemical modifications described herein that modulates the binding affinity between the sense and antisense strands of the siNA construct.

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

hi one embodiment, the invention features siNA constracts that mediate RNAi against Myc and/or Myb, wherein the siNA constract comprises about one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell

In one embodiment, the invention features siNA constracts that mediate RNAi against Myc and/or Myb, wherein the siNA constract comprises about one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target DNA sequence within a cell.

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

hi one embodiment, the invention features siNA constructs that mediate RNAi against Myc and/or Myb, wherein the siNA constract comprises about one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA constract.

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

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

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

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

In one embodiment, the invention features siNA constracts that mediate RNAi against Myc and/or Myb, wherein the siNA construct comprises about one or more chemical modifications described herein that modulates the cellular uptake of the siNA constract.

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

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

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

h another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability, comprising (a) introducing an excipient formulation to a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, and others.

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

In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).

The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to cany out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, prefeπed components of the kit include the siNA and a vehicle that promotes introduction of the siNA. Such a kit can also include instructions to allow a user of the kit to practice the invention.

The term "short interfering nucleic acid", "siNA", "short interfering RNA",

"siRNA", "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule", "siRNA molecule", or "chemically-modified short interfering nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of mediating RNA interference ("RNAi") or gene silencing in a sequence-specific manner; see for example Bass, 2001, Nature, 411, 428-429; Elbashir et al, 2001, Nature, 411, 494-498; Kreutzer et al, hitemational PCT Publication No. WO 00/44895; Zernicka-Goetz et al, hitemational PCT Publication No. WO 01/36646; Fire, hitemational PCT Publication No. WO 99/32619; Plaetinck et al, International PCT Publication No. WO 00/01846; Mello and Fire, hitemational PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; Li et al, hitemational PCT Publication No. WO 00/44914; AUshire, 2002, Science, 297, 1818-1819; Volpe et al, 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; Hall et al, 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al, 2002, RNA, 8, 842-850; Reinhart et al, 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples of siNA molecules of the invention are shown in Figures 4, 5, 6, 12, and Tables III, IV, V, VI, VII and VIII herein. For example the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence coπesponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where one sfrand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand); the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence coπesponding to the target nucleic acid sequence or a portion thereof. Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non- nucleic acid-based linker(s). The siNA can be a polynucleotide with a haiφin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence coπesponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence coπesponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single sfranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence coπesponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5'-phosphate (see for example Martinez et al, 2002, Cell, 110, 563-574 and Schwarz et al, 2002, Molecular Cell, 10, 537-568), or 5',3'- diphosphate. h certain embodiments, the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompass chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2'-hydroxy (2'-OH) containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2'- hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group). Such siNA molecules do not require the presence of ribonucleotides within the siNA molecule to support RNAi; such siNA molecules can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing about one or more nucleotides with 2'-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be refeπed to as short interfering modified oligonucleotides "siMON." As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short haiφin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level, hi a non-limiting example, epigenetic regulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin stracture to alter gene expression (see for example AUshire, 2002, Science, 297, 1818-1819; Volpe et al, 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al, 2002, Science, 297, 2232-2237).

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

By "inhibit" it is meant that the activity of a gene expression product or level of RNAs or equivalent RNAs encoding about one or more gene products is reduced below that observed in the absence of the nucleic acid molecule of the invention, hi one embodiment, inhibition with a siNA molecule preferably is below that level observed in the presence of an inactive or attenuated molecule that is unable to mediate an RNAi response, h another embodiment, inhibition of gene expression with the siNA molecule of the instant invention is greater in the presence of the siNA molecule than in its absence.

By "gene" or "target gene" is meant, a nucleic acid that encodes a RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be ueπveu rrom or contained m any orgamsm, lor example a plant, animal, protozoan, viras, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts.

By "highly conserved sequence region" is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.

By "sense region" is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

By "antisense region" is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence, h addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule.

By "target nucleic acid" is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA.

By "Myc" is meant, any Myc polypeptide, protein and/or polynucleotide encoding a Myc protein (such as polynucleotides refeπed to by Genbank Accession number in Table I or any other Myc transcript derived from a Myc gene).

By "Myc protein" is meant, any Myc peptide or protein or a component thereof, wherein the peptide or protein is encoded by a Myc gene, for example c-Myc, N-Myc, or L-Myc.

By "Myb" is meant, any Myb polypeptide, protein and/or a polynucleotide encoding a Myb protein (such as polynucleotides refeπed to by Genbank Accession number in Table II or any other Myb transcript derived from a Myb gene). By "Myb protein" is meant, any Myb peptide or protein or a component thereof, wherein the peptide or protein is encoded by a Myb gene, for example c-Myb, a-Myb, b- Myb, or v-Myb.

By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al, 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al, 1987, J Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

The siNA molecules of the invention represent a novel therapeutic approach to treat a variety of oncogenic and proliferative diseases and disorders including various cancers including but not limited to multiple drug resistant cancers, such as leulcemias including acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), Acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia; ovarian cancer, breast cancer, cancers of the head and neck, lymphomas such as mantle cell lymphoma, non- Hodgkin's lymphoma, Burkitt's lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, multiple myeloma, melanoma, colorectal cancer, prostate cancer; and proliferative diseases such as restenosis, polycystic kidney disease and any other indications that can respond to the level of Myc and/or Myb in a cell or tissue.

hi one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently about 18 to about 24 nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 17 to about 23 base pairs (e.g., about 17, 18, 19, 20, 21, 22 or 23). In one embodiment, siNA molecules of the invention comprising haiφin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38, 39, 40, 41, 42, 43 or 44) nucleotides in length and comprising about 16 to about 22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs. Exemplary siNA molecules of the invention are shown in Tables III- VIII. Exemplary synthetic siNA molecules of the invention are shown in Tables IV, VI, VII and VIII and/or Figures 4, 5, and 12.

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

The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incoφoration in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables III- VIII and/or Figures 4, 5, and 12. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures. Furthermore, the chemically modified constructs described in Table IX can be applied to any siNA sequence of the invention.

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

By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By

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

By "subject" is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. "Subject" also refers to an organism to which the nucleic acid molecules of the invention can be administered, hi one embodiment, a subject is a mammal or mammalian cells, hi another embodiment, a subject is a human or human cells.

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

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

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

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein (e.g., cancer). For example, to treat a particular disease or condition, the siNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with about one or more drugs under conditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules could be used in combination with about one or more known therapeutic agents to treat a disease or condition. Non-limiting examples of other therapeutic agents that can be readily combined with a siNA molecule of the invention are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.

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

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

hi one embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule refeπed to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in Tables I and II.

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

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

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

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl-on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.

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

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

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

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

Figure 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3'- nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3 '-terminal glyceryl moiety and wherein the two terminal 3 '-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.

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

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

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

RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-

2'-fiuoro modified nucleotides and all purine nucleotides that may be present are 2'-O- methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.

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

Figure 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in Figure 4A-F to a c-Myc siNA sequence.

Figure 6 shows non-limiting examples of different siNA constracts of the invention. The examples shown (constracts 1, 2, and 3) have about 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop stracture shown in construct 2 can comprise a biodegradable linker that results in the formation of constract 1 in vivo and/or in vitro, hi another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA constract 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA constract 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.

Figure 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA haiφin constracts.

Figure 7A: A DNA oligomer is synthesized with a 5 '-restriction site (RI) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined Myc and/or Myb target sequence, wherein the sense region comprises, for example, about

19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.

Figure 7B: The synthetic construct is then extended by DNA polymerase to generate a haiφin stracture having self-complementary sequence that will result in a siNA transcript having specificity for a Myc and/or Myb target sequence and having self- complementary sense and antisense regions.

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

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

Figure 8A: A DNA oligomer is synthesized with a 5 '-restriction (RI) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined

Myc and/or Myb target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3 '-restriction site

(R2) which is adjacent to a loop sequence of defined sequence (X).

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

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

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

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

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

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

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

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

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

Figure 12A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in Figure 4A-F to a c-Myb siNA sequence.

Figure 13 shows a non-limiting example of reduction of Myc (c-Myc) mRNA in 293T cells mediated by chemically-modified siNAs that target c-Myc mRNA. 293T cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. A screen of siNA constructs comprising ribonucleotides and 3 '-terminal dithymidine caps was compared to untreated cells, scrambled siNA control constracts (Scraml and Scram2), and cells transfected with lipid alone (transfection control). As shown in the figure, three of the siNA constructs (RPI 30993/31069; RPI 30995/31071; and RPI 30996/31072) show significant reduction of c-Myc RNA expression.

DETAILED DESCRIPTION OF THE INVENTION

Mechanism of action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically- modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limiting only to siRNA and can be applied to siNA as a whole. By "improved capacity to mediate RNAi" or "improved RNAi activity" is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a, reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention, h this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or a siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced, in vitro and/or in vivo. RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al, 1998, Nature, 391, 806). The coπesponding process in plants is commonly refeπed to as post- transcriptional gene silencing or RNA silencing and is also refeπed to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al, 1999, Trends Genet, 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-sfranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2', 5'-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

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

RNAi has been studied in a variety of systems. Fire et al, 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol, 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al, 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al, 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21 -nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al, 2001, EMBO J., 20, 6877) has revealed certain requirements for siRNA length, stracture, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 -nucleotide siRNA duplexes are most active when containing 3'-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2'-deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3 '-terminal siRNA overhang nucleotides with 2'-deoxy nucleotides (2'-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5 '-end of the siRNA guide sequence rather than the 3 '-end of the guide sequence (Elbashir et al, 2001, EMBO J., 20, 6877). Other studies have indicated that a 5'-phosphate on the target-complementary sfrand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5'-phosphate moiety on the siRNA (Nykanen et al, 2001, Cell, 107, 309).

Synthesis of Nucleic acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs ("small" refers to nucleic acid motifs no more than

100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple stracture of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA stracture. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al, 1992, Methods in Enzymology 211, 3-19, Thompson et al, International PCT Publication No. WO 99/54459, Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incoφorated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2'-O- methylated nucleotides and a 45 sec coupling step for 2'-deoxy nucleotides or 2'-deoxy-2'- fluoro nucleotides. Table X outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M = 6.6 μmol) of 2'-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M = 15 μmol) can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-bound 5'-hydroxyl. A 22-fold excess (40 μL of 0.11 M = 4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M = 10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5'-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltefrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American hitemational Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-l,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

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

The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al, 1987, J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990, Nucleic Acids Res., 18, 5433; and Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al, 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end, and phosphoramidites at the 3 '-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2'-O-methylated nucleotides. Table X outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M = 6.6 μmol) of 2'-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M = 15 μmol) can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-bound 5'- hydroxyl. A 66-fold excess (120 μL of 0.11 M = 13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M = 30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5'- hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-l,2-Benzodithiol-3-one l,l-dioxide0.05 M in acetonitrile) is used.

Deprotection of the RΝA is performed using either a two-pot or one-pot protocol.

For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transfeπed to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65 °C for 10 min. After cooling to -20 °C, the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCΝ:H2O/3:l:l, vortexed and the supernatant is then added to the first supernatant. The combined supematants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyπolidinone, 750 μL TEA and 1 mL TEA-3HF to provide a 1.4 M HF concentration) and heated to 65 °C. After 1.5 h, the oligomer is quenched with 1.5 M NH4HCO3.

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

For purification of the trityl-on oligomers, the quenched NH4HCO3 solution is loaded onto a C-l 8 containing cartridge that had been pre-washed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 min. The cartridge is then washed again with water, salt exchanged with 1 M NaCI and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile. The average stepwise coupling yields are typically >98% (Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.

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

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

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

The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constracts can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al, supra, the totality of which is hereby incoφorated herein by reference) and re-suspended in water. In another aspect of the invention, siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated viras, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.

Optimizing Activity of the nucleic acid molecule of the invention.

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

92/07065; Peπault et al, 1990, Nature 344, 565; Pieken et al, 1991, Science 253, 314;

Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al, hitemational

Publication No. WO 93/15187; Rossi et al, hitemational Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al, U.S. Pat. No. 6,300,074; and Burgin et al, supra; all of which are incoφorated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-0-methyl, 2'-O- allyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al, International Publication PCT No. WO 92/07065; Peπault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat No. 5,334,711 and Beigelman et al, 1995, J Biol. Chem., 270, 25702; Beigelman et al, International PCT publication No. WO 97/26270; Beigelman et al, U.S. Pat. No. 5,716,824; Usman et al, U.S. Pat. No. 5,627,053; Woolf et al, hitemational PCT Publication No. WO 98/13526; Thompson et al, USSN 60/082,404 which was filed on April 20, 1998; Kaφeisky et al, 1998, Tetrahedron Lett, 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al, 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incoφorated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incoφoration of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incoφorated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.

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

Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al, 1995, Nucleic Acids Res. 23, 2677; Carathers et al, 1992, Methods in Enzymology 211, 3-19 (incoφorated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.

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

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

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

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

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

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

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

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

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

In another aspect a siNA molecule of the invention comprises about one or more 5' and/or a 3'- cap structure, for example on only the sense siNA strand, the antisense siNA sfrand, or both siNA strands. By "cap structure" is meant chemical modifications, which have been incoφorated at either terminus of the oligonucleotide (see, for example, Adamic et al, U.S. Pat. No. 5,998,203, incoφorated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5'-terminus (5'-cap) or at the 3'- terminal (3'-cap) or may be present on both termini. In non-limiting examples, the 5'-cap is selected from the group consisting of glyceryl, inverted deoxy abasic residue (moiety); 4',5 '-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; tλreo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5- dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety; 3 '-3 '-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3 '-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'- phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.

In non-limiting examples, the 3'-cap is selected from the group consisting of glyceryl, inverted deoxy abasic residue (moiety), 4',5 '-methylene nucleotide; l-(beta-D- erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; t/zreo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'- inverted abasic moiety; 5'-phosphoramidate; 5 '-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5 '-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incoφorated by reference herein).

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

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

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The prefeπed substiruent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyπolyl, N-lower alkyl pyπolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(0)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.

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

hi one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising about one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, moφholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al, 1994, Novel Backbone Replacements for Oligonucleotides, m Carbohydrate Modifications in Antisense Research, ACS, 24-39.

By "abasic" is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, see for example Adamic et al, U.S. Pat. No. 5,998,203.

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

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

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

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

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to freat for example cancer, restenosis, polycystic kidney disease and other indications that can respond to the level of Myc and/or Myb in a cell or tissue, alone or in combination with other therapies. For example, a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Tlierapeutics, ed. Akhtar, 1995, Maurer et al, 1999, Mol. Membr. Biol, 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol, 137, 165-192; and Lee et al, 2000, ACS Symp. Ser., 752, 184-192, all of which are incoφorated herein by reference. Beigelman et al, U.S. Pat. No. 6,395,713 and Sullivan et al, PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incoφoration into other vehicles, such as hydrogels, cyclodextrins (see for example Gonzalez et al, 1999, Bioconjugate Chem., 10, 1068-1074), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, hitemational PCT Publication No. WO 00/53722). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al, 1999, Clin. Cancer Res., 5, 2330-2337 and Baπy et al, hitemational PCT Publication No. WO 99/31262. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occunence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.

Thus, the invention features a pharmaceutical composition comprising about one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art. The present invention also includes pharmaceutically acceptable formulations ol tήe compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such fonns should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

By "systemic administration" is meant in vivo systemic absoφtion or accumulation of drags in the blood stream followed by distribution throughout the entire body.

Administration routes that lead to systemic absoφtion include, without limitation: intravenous, subcutaneous, infraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drag into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drag to target cells by taldng advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.

By "pharmaceutically acceptable formulation" is meant, a composition or foπnulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non- limiting examples of agents suitable for formulation with the nucleic acid molecules of ^• the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drags into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol, 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, MA); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drags across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery sfrategies for the nucleic acid molecules of the instant invention include material described in Boado et al, 1998, J Pharm. Sci, 87, 1308-1315; Tyler et al, 1999, FEBS Lett, 421, 280-284; Pardridge et al, 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Henada et al, 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al, 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drags in target tissues. This class of drag carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drag (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwaxa et al, Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by exfravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et al, 1995, Biochim. Biophys. Acta, 1238, 86- 90). The long-circulating liposomes enhance the phaπnacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol. Chem. 1995, 42, 24864-24870; Choi et al, hitemational PCT Publication No. WO 96/10391; Ansell et al, hitemational PCT Publication No. WO 96/10390; Holland et al, International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drags from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985), hereby incoφorated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of 7-hydroxybenzoic acid, h addition, antioxidants and suspending agents can be used.

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

The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit fonnulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The teπn parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. About one or more nucleic acid molecules of the invention can be present in association with about one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. compositions intended lor oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain about one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absoφtion in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

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

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

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

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

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

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

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

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

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

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

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water. The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.

h one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol Chem. 262, 4429- 4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). hi another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenaπy or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al, 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J, 4, 317-328, obtained this high specificity through the use of N-acetyl- D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This "clustering effect" has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al, 1981, J. Med. Chem., 24, 1388-1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al, USSN 10/201,394, filed August 13, 2001; and Matulic-Adamic et al, USSN 60/362,016, filed March 6, 2002.

Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weinfraub, 1985, Science, 229, 345; McGaπy and Lindquist, 1986, Proc. Natl. Acad. Sci, USA 83, 399; Scanlon et al,

1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al, 1992, Antisense Res.

Dev., 2, 3-15; Dropulic et al, 1992, J. Virol, 66, 1432-41; Weerasinghe et al, 1991, J. Virol, 65, 5531-4; Ojwang et al, 1992, Proc. Natl. Acad. Sci USA, 89, 10802-6; Chen et al, 1992, Nucleic Acids Res., 20, 4581-9; Sarver et α/., 1990 Science, 247, 1222-1225; Thompson et al, 1995, Nucleic Acids Res., 23, 2259; Good et al, 1997, Gene Tlierapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al, 1991, Nucleic Acids Res., 19, 5125- 30; Ventura et al, 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al, 1994, J Biol. Chem., 269, 25856.

hi another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al, 1996, 77G., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constracted based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or infra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al, 1996, TIG., 12, 510).

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

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

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

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

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

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

Myc biology and biochemistry

Significant progress has been made over the last two decades in our understanding of the function of c-Myc in normal cells and in cancer cells. In contrast to the tightly regulated c-Myc gene in normal cells, which only express the gene when cells actively divide, c-Myc in cancer cells may be expressed in an uncontrolled fashion as the result of genetic abeπations. It is now well established that the deregulated expression of c-Myc plays a significant role in human cancer development. The c-Myc protein or the c-Myc gene is overexpressed in a wide variety of human cancers with 80% of breast cancers, 70% of colon cancers, 90% of gynecological cancers, 50% of hepatocellular carcinomas and a variety of hematological tumors possessing abnormal Myc expression. On the basis of these frequencies, it is estimated that approximately 100,000 U.S. cancer deaths per year are associated with changes in the Myc gene or its expression. The clinical significance of Myc gene alterations in human cancers is best illustrated by prognostication by N-Myc amplification in neuroblastoma, and the sine qua non chromosomal translocation affecting c-Myc in Burkitt's lymphoma. For a recent review of the Myc oncogene, see "Myc Oncogene," The Encyclopedia of Cancer, Second Edition, July 2002, Academic Press.

The c-Myc gene is located on human chromosome 8q24 and comprises three exons.

Transcription of the c-Myc gene can be initiated at one of three promoters. Translation beginning at the AUG start site in the second exon produces a major 439 amino acid protein, the 64 kDa c-Myc protein. Alternative translational initiation start sites result in both longer and shorter forms of the protein, termed p67 Myc and MycS, respectively. The c-Myc protein is O-glycosylated and phosphorylated, and these modifications can alter the protein half-life. The c-Myc sequence contains several conserved N-terminal domains, termed Myc boxes, which are found in closely related proteins, N-Myc and L- Myc.

The C-terminal region of c-Myc contains a dimerization motif, termed the helix- loop-helix leucine zipper (HLH LZ). The HLH LZ domain mediates homotypic or heterotypic dimerization with other HLH LZ proteins. The c-Myc dimerization domain is important for cellular transformation, and the bHLH LZ protein Max has been identified as a c-Myc obligate partner protein. The c-Myc RNA and protein have short half-lives as compared to those of Max, and in most systems Myc appears to be the limiting, regulated component of the heterodimer. Dimerization of Myc and Max through the HLH LZ domain aligns the adjacent basic regions on each molecule to grip onto specific DNA hexanucleotide core sequences, termed E boxes (5'-CA[C/T]GTG-3'). Max can also bind to bHLH LZ Mad family proteins, which can mediate in transcriptional silencing. Mad levels, as opposed to those of Myc, increase during differentiation, and decreased expression of the Mad2 (Mxi-1) protein has been implicated in the development of cancer.

DNA-bound Myc-Max complexes activate transcription through the amino terminal 143 amino acids of c-Myc, termed the transcriptional activation domain (TAD). A small segment of this region is also important for Myc-mediated transcriptional repression. This N-terminal transregulatory domain of c-Myc appears to be required for transformation, and transformation appears to be highly dependent on the region required for Myc mediated transcriptional repression. Both of the activation and repression functions of Myc may be required for neoplastic transformation.

The DNA bound Myc-Max heterodimer interacts through the Myc N-terminal region with a variety of proteins involved in transcription. These include TRRAP, which associates with histone acetylase GCN5. Acetylation of histones may then mark cliromatin to allow access of transcription factors that belong to the general transcriptional machinery, such as TFIIE or TBP, to DNA. Other putative Myc binding proteins have been implicated in regulating Myc transactivating or fransrepressing properties, including pi 07 and Miz-1. The Mad-Max complexes, in contrast to Myc-Max complexes, recrait histone deacetylases that induce compact chromatin structures, which in turn can limit access of transcription factors to DNA.

Expression of c-Myc in the normal cell is tightly regulated by external signals, such as growth factors and extracellular matrix contacts, as well as by internal clocks, such as the cell cycle. The resting cell normally expresses little c-Myc, whereas cells stimulated by growth factors dramatically increase c-Myc expression such that c-Myc can be termed an immediate early response gene. c-Myc expression persists into the cell cycle, but then returns to its basal quiescent state in resultant resting daughter cells. Abnormal or ectopic overexpression of c-Myc in primary cells activates a protective pathway through the induction of pl9/pl4ARF and a p53-dependent cell death pathway. Hence, normal cells that overexpress c-Myc are eliminated from the host organism through apoptosis, thereby protecting the organism from lethal neoplastic changes.

Normal embryonic development appears to require regulated expression of c-Myc as well as other Myc family members. Mouse embryos in which both alleles of c-Myc have been deleted by homologous recombination die in early development with a lack of primitive hematopoiesis. Targeted gene replacement demonstrates that n-Myc can substitute for c-Myc during embryo genesis, indicating that n-Myc functional domains are virtually identical to those of c-Myc. L-Myc null animals have no distinct phenotype, suggesting that 1-Myc is not required for embryogenesis. Embryonic fibroblasts from animals lacking c-Myc show absent or a marked decrease in proliferation rate and viability. The fruitfly Drosophila with mutated Myc alleles are much smaller than wild- type flies, much like the Minute class of small fruitflies, which are mutant in ribosomal protein genes and other yet-unidentified genes. The cells from these mutant flies are small in size, indicating that Myc may participate in the control of normal cell and organismal size. Murine hypomoφhs of c-Myc also cause smaller body size, but here it is due to the decrease in the number of cells rather than smaller cell size.

Activation of the c-Myc gene, which contributes to the development of human cancers, occurs in several ways. Chromosomal translocations, as in the case of Burkitt's lymphoma, activate the c-Myc locus by juxtaposing it adjacent to immunoglobulin genes that are transcriptionally highly active in B cells. Gene amplification increases Myc gene copy number, which in turn increases Myc expression. This is most dramatically found in other members of the Myc family, where over 200 copies per cell of N-Myc can be found in neuroblastoma, and over 50 copies per cell of c-Myc, N-Myc, or L-Myc may be found in small cell lung cancers. Increased c-Myc gene transcription can account for the observed elevation of Myc in human colon carcinoma. Recent studies suggest that the transcriptional co-activator a-catenin, which is inactivated when bound to the adenomatous polyposis coli (APC) protein, might activate Myc expression in the absence of APC through binding the TCF4 transcription factor. Mutant, activated a-catenin proteins mat are lound m colon, liver and other cancers may constitutively deregulate Myc expression leading to the development of these cancers.

Other mechanisms of c-Myc overexpression include removal of 3' UTR destabilizing sequences, resulting in an elevation of Myc mRNA. Insertion of retrovimses adjacent to the Myc locus activates its expression via retroviral regulatory sequences. Oncogenic ras appears to stabilize the Myc protein through a hypothesized post-translational modification mechanism.

A variety of models have been utilized to demonstrate that c-Myc is oncogenic. When immortalized Rat la fibroblasts are engineered to overexpress c-Myc, they acquire the ability to grow in soft agar, signifying loss of contact inhibition commonly found in tumor cells. Furthermore, transgenic animals with tissue-targeted expression of c-Myc form tumors in the targeted tissues. More recently it has been shown that the conditional induction of c-Myc transgene in vivo in keratinocytes, mammary epithelial cells or hematopoietic cells can lead to reversible proliferation and clonal expansion, a hallmark of neoplasia.

Although a link between c-Myc and cancer is well established both in vivo and in vitro, recent work has established a role for c-Myc in cell cycle progression, metabolism, apoptosis and genomic instability. c-Myc is thought to promote cell proliferation and genomic instability by accelerating cells through GI and S phases of the cell cycle, abrogating cell cycle checkpoints, and increasing cell metabolism. In many settings these alterations can lead to apoptosis, or cell death. However, in the background of additional mutations that activate anti-apoptotic signals, c-Myc can lead to complete neoplastic transformation.

Recent efforts have concentrated on identifying the genetic program induced by c- Myc. Direct targets of c-Myc are defined as those target genes each of whose expression is directly altered by direct c-Myc binding to each genes, respectively. Indirect targets are those activated by genes that are direct targets of Myc (i.e. induced by transcription factors, whose genes are direct c-Myc targets). c-Myc target genes have been identified with several techniques that compare the expression of genes in cell lines that overexpress c-Myc with ones that do not. With the availability of cells that are depleted of c-Myc through homologous recombination, it is now more feasible to study the role of c-Myc in normal cell homeostasis. It is important, however, to distinguish between c-Myc targets induced in normal cell homeostasis versus those induced in pathophysiological, neoplastic conditions in which c-Myc is overexpressed. Myc target genes have been identified in a variety of ways, including by DNA microaπay analysis and SAGE. While none of the Myc responsive genes by itself recapitulates the complete Myc phenotype, many appear necessary and hence have been implicated in Myc functions, such as apoptosis, cell cycle control, and cell adhesion.

The removal of c-Myc by homologous recombination results in cells that have remarkably slowed population growth doubling time. This slowed growth, however, appears to result in the decreased probability of a cell to enter into the cell cycle in the absence of c-Myc. As a result, only a small fraction of the Myc null cell population enters the cell cycle with a virtually normal cell doubling time. This population multiplies for several cycles and then returns to a resting phase as other cell populations enter the cell cycle. These observations, which are based on single cell studies, suggest that c-Myc controls the probability of a cell to enter the cell cycle. The challenge is to understand how c-Myc affects the stochastic nature of cell proliferation at the molecular level. Most studies have focused on c-Myc 's effect on regulatory proteins of the Gl-S phase transition of the cell cycle. This transition is promoted when cyclin dependent kinases (CDKs) are activated by association with specific cyclins. Cyclin dependent kinase inhibitors (CDKIs) inhibit this activation. c-Myc has been implicated in inducing cyclin DI and D2, cyclin E, CDK4, and cdc25A, a phosphatase which activates CDK2 and CDK4. c-Myc has also been shown to lower the amounts or inhibit the function of the CDK inhibitor, p27, potentially by increasing cyclin D levels which can then sequester p27. c-Myc also induces Cull, which mediates the degradation of p27.

A highly regulated cell cycle permits cells to repair DNA damage before replicating, thus promoting genomic fidelity. Inappropriate cell cycle proliferation can lead to genomic instability, resulting in new mutations and abnormal chromosome number and stracture. c-Myc overexpression, even transiently, can induce genomic instability that is characterized by gene amplification, aneuploidy and polyploidy. Other studies suggest that c-Myc induces the production of reactive oxygen species (ROS) by mitochondria, leading to DNA damage and genomic instability. Burkitt's lymphoma has long been noted by pathologists to have not only a high mitotic index, but also by a high apoptotic rate as illustrated by the abundance of abnormal nuclei. c-Myc induced apoptosis was first recognized in studies of the IL-3 dependent 32d.3 myeloid progenitor cell line. In the absence of IL-3, enforced c-Myc expression drives cells into S phase and accelerates the rate of cell death. Fibroblasts that overexpress c-Myc also undergo apoptosis in response to environmental stresses, including low serum, hypoxia, and low glucose. The regions of Myc that are required for apoptosis are also those that are required for cellular transformation. Although several Myc target genes, such as omithine decarboxylase (ODC) and lactate dehydrogenase A (LDH-A), appear to be involved in Myc-induced apoptosis, the mechanism of Myc action in apoptosis is not clear. In the case of primary cells, pl9/pl4ARF is likely to be a relevant downstream effector of Myc-induced apoptosis. Myc also activates the telomerase hTERT, which encodes an enzyme that sustains telomere length and leads to immortalization of cells. The relevant clinical coπelation is the intriguing observation that both N-Myc amplification and telomerase levels are parallel predictors of poor outcome in neuroblastoma. This observation can be construed to suggest that N-Myc is responsible for the elevation of telomerase in neuroblastoma.

Proliferation requires increased energy and availability of biosynthetic substrates, and would therefore necessitate increase metabolic rates. It is well established that the major component of a eukaryotic cell is its ribosomal mass, which constitutes up to 40% of total mass and accounts for 90%) of cellular RNA mass. It is also notable that as a cell traverses through the GI phase of the cell cycle, its mass gradually increases prior to the initiation of DNA synthesis in S phase. The regulation of cell mass is linked to the cell cycle, although the molecular basis for this regulation is largely unknown, h triguingly, mutation of the Drosophila Myc gene results in small flies that resemble flies that bear mutant ribosomal protein genes. These small flies are small not because they have fewer cells, but rather their cells are smaller in size. These observations suggest that Myc participates in the regulation of cell mass, and this notion has gained support from observations in specific mammalian cells that overexpress c-Myc. Recent studies have suggested that c-Myc overexpression results in elevated expression of genes encoding translational factors and ribosomal proteins, which in turn may contribute to increased cellular mass. ±sotή large and small tumors are often hypoxic as a result of diminished, disordered neovascularization. Survival and rapid proliferation of a tumor in a hostile environment is often accompanied by a switch of cancer cells to use glucose as an energy source without oxygen and leads to an oveφroduction of lactic acid. The transcription factor, HIF-1, is induced by hypoxia and transactivates many glycolytic enzymes necessary for this switch. Interestingly, HIF-1 binds to a DNA motif in the promoter of these genes that is very similar to the Myc E-box. Myc is able to transactivate many of these same glycolytic genes and the glucose transporter GLUT-1. Hence, the widespread deregulation of c-Myc in human cancers may contribute to enhanced tumor glycolysis, known as the Warburg effect. Recently, c-Myc was shown to regulate a nuclear gene encoding a mitochondrial peroxiredoxin, which appears required for cell proliferation and transformation mediated by c-Myc. Furthermore, the mitochondrial serine hydroxymethylfransferase, which is involved in single carbon metabolism, was able to partially rescue the growth of rat Myc null cells in an expression cloning strategy. Hence, the pleiotropic effects of c-Myc on cellular metabolism and cell growth remain to be clearly delineated.

As cells prepare for S phase, adequate substrates must accumulate to ensure the fidelity of DNA replication. Genes that are important in nucleotide biosynthesis and DNA metabolism are also implicated as Myc targets, such as carbamoyl phosphate synthase (CAD), ODC, dihydrofolate reductase and thymidine kinase. Although little is known about the possible role of c-Myc in regulating cellular metabolism during the second cell cycle gap, G2, and during mitosis, a complex role for c-Myc in regulating cellular metabolism has clearly emerged from recent studies.

Based upon the cuπent understanding of Myc biology and biochemistry, the modulation of c-Myc and other genes involved in the Myc pathway is instrumental in the development of new therapeutics in the field of oncology, including in related fields of cell cycle regulation, apoptosis, metabolism, cell adhesion, and cell differentiation. As such, modulation of Myc genes and other genes involved in the Myc pathway using small interfering nucleic acid (siNA) mediated RNAi represents a novel approach to the treatment, diagnosis, and study of diseases and conditions related to Myc activity and/or gene expression, including cancer and other proliferative disorders, such as restenosis. Myb biology and biochemistry

The c-myb proto-oncogene is the cellular homolog of the avian myeloblastosis viras transforming gene v-myb. The c-myb protein is a transcription factor involved in the regulation of hematopoietic cell proliferation and differentiation. C-myb is highly expressed in primitive hematopoietic cells, and its expression decreases during maturation and differentiation of hematopoietic cells. C-myb expression is also elevated in some primary hematopoietic tumors and in some leukemic cell lines. The in vifro and in vivo expression of c-myb can result in transformation of preferentially myeloid lineage hematopoietic cells. Since the myb gene encodes proteins that are critical for hematopoietic cell proliferation and development, disrupting myb function has been proposed as an effective therapeutic strategy for controlling leukemic cell growth. An animal model for testing the in vivo efficacy of antisense oligodeoxynucleotides targeting myb mRNA has been developed (Ratajczak et al, 1992, Proc. Nat Acad. Sci, 89, 11823- 11827). This model comprises a human leukemia-scid mouse chimera with K562 cells derived from a patient with chronic myelogenous leukemia. Mice treated with the antisense targeting myb expression survived at least 3.5 times longer than control animals. In addition, animals receiving antisense myb DNA had significantly less disease at the two sites most frequently involved by leukemic cell infiltration, namely the CNS and the ovary.

The myb gene is also implicated in restenosis following angioplasty. When whole serum is added to serum-starved smooth muscle cells in vitro, the oncogenes c-myc, c- fos, and c-myb, are induced (Kindy and Sonenshein, 1986, J. Biol. Chem., 261, 12865- 12868) and cell proliferation ensues. Blocking c-myb with an antisense oligonucleotide prevents cells from entering S phase (Brown, K. E., et al, 1992, J. Biol. Chem., 267, 4625-4630.). Thus, c-myb is thought to be required for the Gl to S transition after stimulation by the multitude of growth factors present in serum. In vivo, a c-myb antisense oligonucleotide inhibits restenosis when applied to rat arteries after balloon angioplasty (Simons, M., et al, 1992, Nature, 359, 67-70). Similarly, an antisense oligonucleotide directed against mRNA of the oncogene c-Myc was shown to inhibit human smooth muscle cell proliferation (Shi, Y., et al, 1993, Circulation, 88, 1190-5) and migration (Biro, S., et al, 1993, Proc. Natl. Acad. Sci. USA, 90, 654-8). Therefore, the use of small interfering nucleic acid molecules targeting cellular oncogenes, such as c-myb, and genes involved in the c-myb expression pathway, such as IL-2, bcl-2, protein kinase B (PKB), phosphoinositide 3-kinase (PI3K), E2F, NFkappaB, c-myc, and pim-1, provides a class of novel therapeutic agents that can be used in the treatment of various cancers, such as leukemia and proliferative conditions such as restenosis.

Examples:

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

Example 1: Tandem synthesis of siNA constracts

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

After completing a tandem synthesis of a siNA oligo and its complement in which the 5'-terminal dimethoxytrityl (5'-O-DMT) group remains intact (trityl-on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence sfrands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one sfrand has retained the 5'-O-DMT group while the complementary strand comprises a terminal 5 '-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see Figure 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamme (DIP A) and/or DMAP in the presence of an activator reagent such as Bromolripyπolidinophosphoniunihexaflurorophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5'-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50mM NaOAc or 1.5M NH4H2CO3.

Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example using a Waters C18 SepPak lg cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2 CV 50mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50mM NaOAc and 50mM NaCI). The column is then washed, for example with 1 CV H2O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H20 followed by 1 CV IM NaCI and additional H2O. The siNA duplex product is then eluted, for example using 1 CV 20% aqueous CAN.

Figure 2 provides an example of MALDI-TOV mass spectrometry analysis of a purified siNA constract in which each peak coπesponds to the calculated mass of an individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably coπesponding to the duplex siNA, and two peaks presumably coπesponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract only shows a single peak. Testing of the purified siNA constract using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constracts generated from separately synthesized oligonucleotide sequence strands. Example 2: Identification of potential siNA target sites in any RNA sequence

The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA stracture, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non- limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA constract to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.

Example 3: Selection of siNA molecule target sites in a RNA

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

1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well. 2. hi some instances the siNAs coπespond to more than one target sequence; such would be the case for example in targeting different franscripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify sub sequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.

3. In some instances the siNA subsequences are absent in about one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and ranked according to self- folding and internal hafrpins. Weaker internal folds are prefeπed; strong haiφin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense sfrand. 7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3'-end of the sequence, and/or AA on the 5'-end of the sequence (to yield 3' UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.

8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) sfrand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Tables III- III). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3' terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most prefeπed target site within the target RNA sequence.

In an alternate approach, a pool of siNA constracts specific to a Myc and/or Myb target sequence is used to screen for target sites in cells expressing Myc and/or Myb RNA, such as human aortic smooth muscle cells (e.g., HASMC) for Myb RNA and HeLa cells for Myc RNA. The general strategy used in this approach is shown in Figure 9. A non-limiting example of such a pool is a pool comprising sequences having sense sequences comprising SEQ ID NOs. 1-118, 595-598, 606-609, 614-617, 622-625, 237- 415, 599-602, 630-633, 636-639, and 644-647, and antisense sequences comprising SEQ JD NOs. 119-236, 610-613, 618-621, 626-629, 416-594, 603, 604, 634, 635, 640-643, and 648-651, respectively. Cells expressing Myc and/or Myb are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with Myc and/or Myb inhibition are sorted. The pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example Figure 7 and Figure 8). The siNA from cells demonsfrating a positive phenotypic change (e.g., decreased proliferation, decreased Myc and/or Myb mRNA levels or decreased Myc and/or Myb piυiem expression , are sequenced to determine tne most suitable target site(s) within the target Myc and/or Myb RNA sequence.

Example 4: Myc and/or Mvb targeted siNA design

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

Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constracts are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g., liver extracts). The synthetic siNA constracts are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constracts that possess both nuclease stability and RNAi activity can be further modified and re- evaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for example Figure 11). Example 5: Chemical Synthesis and Purification of siNA

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

In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5'-O- dimethoxytrityl, 2'-O-tert-butyldimethylsilyl, 3'-O-2-Cyanoethyl N,N-diisopropylphos- phoroamidite groups, and exocyclic amine protecting groups (e.g., N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2'-O-Silyl Ethers can be used in conjunction with acid-labile 2 '-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2' chemistries can require different protecting groups, for example 2 '-deoxy-2 '-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al, US Patent 5,631,360, incoφorated by reference herein in its entirety).

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

Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in Scaringe supra, Usman et al, US 5,831,071, US 6,353,098, US 6,437,117, and Bellon et al, US 6,054,576, US 6,162,909, US 6,303,773, herein incoφorated by reference in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constracts. For example, applicant has observed that oligonucleotides comprising 2'-deoxy-2'-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35°C for 30 minutes. If the 2 '-deoxy-2 '-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35°C for 30 minutes, TEA-HF is added and the reaction maintained at about 65°C for an additional 15 minutes.

Example 6: RNAi in vitro assay to assess siNA activity

An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting Myc and/or Myb RNA targets. The assay comprises the system described by Tuschl et al, 1999, Genes and Development, 13, 3191-3197 and Zamore et al, 2000, Cell, 101, 25-33 adapted for use with Myc and/or Myb 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 Myc and/or Myb expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES- KOH, pH 7.4, 2 mM magnesium acetate) for 1 min. at 90°C followed by 1 hour at 37°C, then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES- KOH at pH 7.4, 2mM 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 dechlorinated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concenfration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug.ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C for 10 minutes before adding RNA, then incubated at 25° C for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25 x Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-³²p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5'-32p-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites the Myc and/or Myb

RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constracts are screened for RNAi mediated cleavage of the Myc and/or Myb RNA target, for example by analyzing the assay reaction by elecfrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.

Example 7: Nucleic acid inhibition of Myc and/or Myb target RNA in vivo siNA molecules targeted to the human Myc and/or Myb RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the Myc RNA are given in Tables III, IV and VII, and the target sequences and the nucleotide location within the Myb RNA are given in Tables V, VI and VIII.

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

Delivery of siNA to Cells

Cells (e.g., HeLa, HASMC) are seeded, for example, at 1x10^ cells per well of a six-well dish in EGM-2 (BioWhittaker) the day before transfection. siNA (final concentration, for example 20nM) and cationic lipid (e.g., final concentration 2μg/ml) are complexed in EGM basal media (Biowhittaker) at 37°C for 30 minutes in polystyrene tubes. Following vortexing, the complexed siNA is added to each well and incubated for the times indicated. For initial optimization experiments, cells are seeded, for example, at lxl 0^ in 96 well plates and siNA complex added as described. Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid. Cells in 6- well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope. Taqman and Lightcycler quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example, using Qiagen RNA purification kits for 6-well or Rneasy exfraction kits for 96-well assays. For Taqman analysis, dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5'-end and the quencher dye TAMRA conjugated to the 3 '-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, IX TaqMan PCR reaction buffer (PE- Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AmphTaq Gold (PE-Applied Biosystems) and 10U M- MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 min at 48°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 minute at 60°C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/rxn) and normalizing to β-actin or GAPDH mRNA in parallel TaqMan reactions. For each gene of interest an upper and lower primer and a fluorescently labeled probe are designed. Real time incoφoration of SYBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler. A standard curve is generated for each primer pair using control cRNA. Values can be represented as relative expression to GAPDH in each sample.

Western blotting

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

Example 8: Models useful to evaluate the down-regulation of Myc gene expression

Cell Culture

There are numerous cell culture systems that can be used to analyze reduction of

Myc levels either directly or indirectly by measuring downstream effects. For example, cultured HeLa cells can be used in cell culture experiments to assess the efficacy of nucleic acid molecules of the invention. As such, HeLa cells treated with nucleic acid molecules of the invention (e.g., siNA) targeting Myc RNA would be expected to have decreased Myc expression capacity compared to matched confrol nucleic acid molecules having a scrambled or inactive sequence, hi a non-limiting example, HeLa cells are cultured and Myc expression is quantified, for example by time-resolved immuno fluorometric assay. Myc messenger-RNA expression is quantitated with RT-PCR in cultured HeLas. Untreated cells are compared to cells treated with siNA molecules transfected with a suitable reagent, for example a cationic lipid such as lipofectamine, and Myc protein and RNA levels are quantitated. Dose response assays are then performed to establish dose dependent inhibition of Myc expression.

In several cell culture systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, et al, 1992, Mol. Pharmacology, 41, 1023-1033). h one embodiment, siNA molecules of the invention are complexed with cationic lipids for cell culture experiments. siNA and cationic lipid mixtures are prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives are warmed to room temperature (about 20-25 °C) and cationic lipid is added to the final desired concentration and the solution is vortexed briefly. siNA molecules are added to the final desired concentration and the solution is again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex is serially diluted into DMEM following the 10 minute incubation. Animal Models

Evaluating the efficacy of anti-Myc agents in animal models is an important prerequisite to human clinical trials. Barisoni et al, 1995, Am J Pathol, 147, 1728-35 describe a transgenic mouse model of polycystic kidney disease. Adult polycystic kidney disease is believed to be the most frequent (1/500) inherited genetic disorder in humans. Barisoni et al, supra generated a genetic model of the disease in transgenic mice by introducing a deregulated proto-oncogene c-myc specifically expressed in the kidney. All transgenic lines produced develop adult polycystic kidney disease in a reproducible manner. The clinical phenotype observed in mice is present at birth and leads to renal insufficiency in adulthood. The investigators determined that abnormal proliferation and programmed cell death are responsible for cystogenesis in polycystic kidney disease. Furthermore, this phenomena is controlled by a specific c-myc mechanism independent of the p53 pathway. A similar mechanism also prevails in human autosomal dominant polycystic kidney disease. Therefore, this murine model provides a useful model to understand the polycystic kidney disease pathogenesis and can be used to evaluate potential therapeutic agents such as siNA molecules of the invention.

Other animal models known in the art can be used to evaluate siNA molecules of the invention targeting c-Myc for other disease conditions, see for example Schmidt et al, 1988. PNAS USA., 85:6047-6051 (describing transgenic mice bearing the human c-myc gene activated by an immunoglobulin enhancer useful as a pre-B-cell lymphoma model) and Hoyer et al., 2002, PNAS USA., 99, 14392-97 (describing transgenic mice that develop Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma (DLBCL)). Animals freated with siNA molecules of the invention targeting Myc RNA can be evaluated for clinical response (e.g., decreased tumor size/metastasis) and/or decreased levels of Myc RNA or protein.

Example 9: Models useful to evaluate the down-regulation of Myb gene expression

Cell Culture

Rat vascular smooth muscle cells (RASMC) are an example of cells that can be used to analyze reduction of Myb levels either directly or indirectly by measuring downstream effects. Rat vascular smooth muscle cells are isolated and cultured as follows. Aortas from adult Sprague-Dawley rats are dissected, connective tissue is removed under a dissecting microscope, and 1 mm² pieces of the vessel are placed, intimal side up, in a Petri dish in Modified Eagle's Medium (MEM) with the following additives: 10% FBS, 2% tryptose phosphate broth, 1% penicillin streptomycin and 2 mM L-Glutamine. The smooth muscle cells are allowed to migrate and grow to confluence over a 3-4 week period. These primary cells are frozen and subsequent passages are grown at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), and the following additives: 2 mM L-Glutamine, 1% penicillin/streptomycin, 1 mM sodium pyravate, non-essential amino acids (0.1 mM of each amino acid), and 20 mM Hepes pH 7.4. Cells passed four to six times are used in proliferation assays. For the cell proliferation assays, 24-well tissue culture plates are prepared by coating the wells with 0.2%) gelatin and washing once with phosphate- buffered saline (PBS). RASMC are inoculated at lxlO⁴ cells per well in 1 ml of DMEM plus 10%) FBS and additives and incubated for 24 hours. The cells become sub confluent when plated at this density. The cells are serum-starved by removing the medium, washing once with PBS, and incubating 48-72 hours in DMEM containing 0.5% FBS plus additives.

h several other systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, et al, 1992, Mol. Pharmacology, 41, 1023-1033). hi one embodiment, siNA molecules of the invention are complexed with cationic lipids for cell culture experiments. siNA and cationic lipid mixtures are prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives are warmed to room temperature (about 20-25 °C) and cationic lipid is added to the final desired concentration and the solution is vortexed briefly. siNA molecules are added to the final desired concentration and the solution is again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex is serially diluted into DMEM following the 10 minute incubation.

Serum-starved smooth muscle cells are washed twice with PBS, and the siNA/lipid complex is added. The plates are incubated for 4 hours at 37°C. The medium is then removed and DMEM containing 10% FBS, additives and 10 μM bromodeoxyuridine (BrdU) is added, hi some wells, FBS is omitted to determine the baseline of unstimulated proliferation. The plates are incubated at 37°C for 20-24 hours, fixed with 0.3%) hydrogen peroxide in 100% methanol, and stained for BrdU incoφoration by standard methods, hi this procedure, cells that have proliferated and incoφorated BrdU stain brown; non-proliferating cells are counter-stained a light puφle. Both BrdU positive and BrdU negative cells are counted under the microscope. The percentage of the total cells that have incoφorated BrdU (% cell proliferation) is determined. Percent inhibition is calculated from the % cell proliferation values as follows: % inliibition = 100 - 100[(siNA - 0% serum)/(Control - 0% serum)]. As such, the efficacy of siNA molecules of the invention targeting Myb can be determined via inhibition of smooth muscle cell proliferation.

Animal Models

Evaluating the efficacy of anti-Myb agents in animal models is an important prerequisite to human clinical trials. Smooth muscle cell (SMC) proliferation is an important component of restenosis in response to injury after balloon angioplasty. The rat carotid artery balloon injury model is a well-characterized and highly reproducible vascular proliferative disorder that is dependent on SMC migration and proliferation. Accordingly, this model can be used to determine whether the inhibition of SMC proliferation by siNA observed in cell culture experiments is sufficient to impact neointima formation in a rat carotid artery model of balloon angioplasty. Rat carotid arteries are subjected to balloon angioplasty and immediately exposed to siNA or inactive control siNA molecules. In a non-limiting example, Sprague-Dawley rats are subjected to balloon angioplasty of the left common carotid artery by dilatation with a Fogarty catheter. Immediately following injury, siNA, or inactive confrol siNA molecules in a volume of 50 to 100 μl is instilled into a 1-cm segment of the distal common carotid artery for 5 minutes by using a 24-gauge intravenous catheter. Rat carotid arteries are harvested 20 days after balloon injury and adenovirus infection. Tissue sections are stained with hematoxylin and eosin. Intimal and medial boundaries are determined by digital planimetry of tissue sections. Areas and ratios are determined from four to six stained sections of each artery spanning the 1-cm site of balloon injury. As such, the efficacy of siNA molecules of the invention can be determined via inhibition of smooth muscle cell proliferation in the rat carotid artery balloon injury model. .e mple m: IUNAI meαiaτed lnmoition oi Myc KJ A expression

siNA constracts were tested for efficacy in reducing Myc (c-Myc) RNA expression in 293T cells. 293T cells were plated approximately 24h before fransfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells were 70-90% confluent. For fransfection, annealed siNAs were mixed with the fransfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 min. at room temperature. The siNA transfection mixtures were added to cells to give a final siNA concenfration of 25 nM in a volume of 150 μl. Each siNA transfection mixture was added to 3 wells for triplicate siNA treatments. Cells were incubated at 37°C for 24h in the continued presence of the siNA transfection mixture. At 24h, RNA was prepared from each well of freated cells. The supematants with the transfection mixtures were first removed and discarded, then the cells were lysed and RNA prepared from each well. Target gene expression following treatment was evaluated by RT-PCR for the target gene and for a confrol gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data were averaged and the standard deviations determined for each treatment. Normalized data were graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs was determined.

Results of this study are shown in Figure 13. A screen of siNA constracts was compared to untreated cells, scrambled siNA control constructs (Scraml and Scram2), and cells transfected with lipid alone (fransfection control). As shown in the figure, three of the siNA constructs (RPI 30993/31069; RPI 30995/31071; and RPI 30996/31072) show significant reduction of c-Myc RNA expression. Additional stabilization chemistries as described in Table LX are similarly assayed for activity.

Example 11 : Indications

The present body of knowledge in Myc and Myb research indicates the need for methods and compounds that can regulate Myc and/or Myb gene product expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used to treat leukemias including acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), Acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia; ovarian cancer, breast cancer, cancers of the head and neck, lymphomas such as mantle cell lymphoma, non-Hodgkin's lymphoma, and Burkitt's lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, multiple myeloma, melanoma, colorectal cancer, prostate cancer, and proliferative diseases such as restenosis, polycystic kidney disease and any other diseases or conditions that are related to or will respond to the levels of Myc and/or Myb in a cell or tissue, alone or in combination with other therapies.

The use of radiation treatments and chemotherapeutics, such as Gemcytabine and cyclophosphamide, are non-limiting examples of chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g., siNA molecules) of the instant invention. Those skilled in the art will recognize that other anti- cancer and/or antiproliferative compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g., siNA molecules) and are hence within the scope of the instant invention. Such compounds and therapies are well known in the art (see for example Cancer: Principles and Practice of Oncology, Volumes 1 and 2, eds Devita, V.T., Hellman, S., and Rosenberg, S.A., J.B. Lippincott Company, Philadelphia, USA; incoφorated herein by reference) and include, without limitations, folates, antifolates, pyrimidine analogs, fluoropyrimidines, purine analogs, adenosine analogs, topoisomerase I inhibitors, anthrapyrazoles, retinoids, antibiotics, anthacyclins, platinum analogs, alkylating agents, nitrosoureas, plant derived compounds such as vinca alkaloids, epipodophyllotoxins, tyrosine kinase inhibitors, taxols, radiation therapy, surgery, nutritional supplements, gene therapy, radiotherapy, for example 3D- CRT, immunotoxin therapy, for example ricin, and monoclonal antibodies. Specific examples of chemotherapeutic compounds that can be combined with or used in conjunction with the nucleic acid molecules of the invention include, but are not limited to, Paclitaxel; Docetaxel; Methofrexate; Doxorubin; Edatrexate; Vinorelbine; Tamoxifen; Leucovorin; 5-fluoro uridine (5-FU); Ionotecan; Cisplatin; Carboplatin; Amsacrine; Cytarabine; Bleomycin; Mitomycin C; Dactinomycin; Mithramycin; Hexamethyhnelamine; Dacarbazine; L-asperginase; Nifrogen mustard; Melphalan, Chlorambucil; Busulfan; Ifosfamide; 4-hydroperoxycyclophosphamide, Thiotepa; Irinotecan (CAMPTOSAR®, CPT-11, Camptothecin-11, Campto) Tamoxifen, Herceptin; IMC C225; ABX-EGF: and combinations thereof. The above list provides non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g., siNA) of the instant invention. Those skilled in the art will recognize that other drag compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g., siNA molecules) are hence within the scope of the instant invention.

Example 12: Diagnostic uses

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

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

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

One skilled in the art would readily appreciate that the present invention is well adapted to cany 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 prefeπed embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constracts with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying siNA molecules with improved RNAi activity.

The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by prefeπed 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.

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

Table II: Myb Accession Numbers

Table III: Myc siNA and Target Sequences

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

\j\ji\j ι y)

Table IV: Myc Synthetic Modified siNA constructs

uppercase = ribonucleotide ,c = 2'-deoxy-2'-fluoro U, C = thymidine = inverted deoxy abasic = phosphorothioate linkage = deoxy Adenosine ' = deoxy Guanosine

Table V: Myb siNA and Target Sequences

Table VI: Myb Synthetic Modified siNA constructs

Uppercase = ribonucleotide u,c = 2'-deoxy-2'-fluoro U, C T = thymidine B = inverted deoxy abasic s = phosphorothioate linkage A - deoxy Adenosine G = deoxy Guanosine

Table VTI: Myc Synthetic Modified siNA constructs

Uppercase = ribonucleotide u,c = 2'-deoxy-2'-fluoro U, C

T = thymidine

B = inverted deoxy abasic s = phosphorothioate linkage

A — deoxy Adenosine

G = deoxy Guanosine

Table VIII: Myb Synthetic Modified siNA constructs

Uppercase = ribonucleotide u,c = 2'-deoxy-2'-fluoro U, C T = thymidine B = inverted deoxy abasic s = phosphorothioate linkage

Table IX

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

Cap = any terminal cap, see for example Figure 10.

All Stab 1-11 chemistries can comprise 3 '-terminal thymidine (TT) residues

All Stab 1-11 chemistries typically comprise 21 nucleotides, but can vary as described herein.

S = sense strand

AS = antisense strand

Table X

A. 2.5 μmol Synthesis Cycle ABI 394 Instrument

B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

C. 0.2 μmol Synthesis Cycle 96 well Instrument

Wait time does not include contact time during delivery.

Tandem synthesis utilizes double coupling of linker molecule

Claims

CLAIMSWhat we claim is:

1. A short interfering nucleic acid (siNA) molecule that down-regulates expression of one or more Myc genes by RNA interference.

2. The siNA molecule of claim 1, wherein the Myc gene is c-Myc.

3. The siNA molecule of claim 1, wherein said siNA molecule comprises no ribonucleotides.

4. The siNA molecule of claim 1, wherein said siNA molecule comprises ribonucleotides.

5. The siNA molecule of claim 1, wherein said siNA molecule is double stranded.

6. The siNA molecule of claim 5, wherein said siNA molecule comprises an antisense strand including nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a Myc protein, and wherein said siNA molecule further comprises a sense strand, wherein said sense strand comprises nucleotide sequence corresponding to nucleotide sequence of a Myc gene or a portion thereof.

7. The siNA molecule of claim 6, wherein said antisense strand and said sense strand each comprise about 19 to about 29 nucleotides, and wherein said antisense strand and said sense strand share at least about 19 complementary nucleotides.

8. The siNA molecule of claim 5, wherein said siNA molecule comprises an antisense region including nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a Myc protein, and wherein said siNA molecule further comprises a sense region, wherein said sense region comprises nucleotide sequence corresponding to nucleotide sequence of a Myc gene or a portion thereof.

9. The siNA molecule of claim 8, wherein said antisense region and said sense region each comprise about 19 to about 29 nucleotides, and wherein said antisense region and said sense region share at least about 19 complementary nucleotides.

10. The siNA molecule of claim 1, wherein said siNA molecule is single stranded.

11. The siNA molecule of claim 10, wherein said siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a Myc protein.

12. The siNA molecule of claim 11, wherein said siNA molecule comprises a sequence having about 19 to about 29 nucleotides.

13. The siNA molecule of claim 1, wherein said siNA molecule comprises a sense region and an antisense region and wherein said antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a Myc protein and said sense region comprises sequence complementary to said antisense region.

14. The siNA molecule of claim 1, wherein said siNA molecule is assembled from two oligonucleotide fragments, wherein one oligonucleotide fragment comprises a sense region and a second oligonucleotide fragment comprises an antisense region of said siNA molecule.

15. The siNA molecule of claim 13, wherein said sense region and said antisense region comprise separate oligonucleotides.

16. The siNA molecule of claim 13, wherein said sense region and said antisense region are connected via a linker molecule.

17. The siNA molecule of claim 16, wherein said linker molecule is a polynucleotide linker.

18. The siNA molecule of claim 16, wherein said linker molecule is a non-nucleotide linker.

19. The siNA molecule of claim 13, wherein said sense region comprises a 3'-terminal overhang and said antisense region comprises a 3'-teπninal overhang.

20. The siNA molecule of claim 19, wherein said 3'-terminal overhangs each comprise about 2 nucleotides.

21. The siNA molecule of claim 19, wherein said antisense region 3 '-terminal overhang is complementary to RNA encoding a Myc protein.

22. The siNA molecule of claim 13, wherein said sense region comprises one or more 2'-O-methyl pyrimidine nucleotides and one or more 2'-deoxy purine nucleotides.

23. The siNA molecule of claim 13, wherein any pyrimidine nucleotides present in said sense region comprise 2'-deoxy-2'-fluoro pyrimidine nucleotides and wherein any purine nucleotides present in said sense region comprise 2'-deoxy purine nucleotides.

24. The siNA molecule of claim 19, wherein any nucleotides comprising said 3 '-terminal nucleotide overhang that are present in said sense region are 2'-deoxy nucleotides.

25. The siNA molecule of claim 13, wherein said sense region comprises a 3 '-end and a 5'-end, and wherein a terminal cap moiety is present at the 5'-end, the 3'-end, or both of the 5' and 3' ends of said sense region.

26. The siNA molecule of claim 25, wherein said terminal cap moiety is an inverted deoxy abasic moiety.

27. The siNA molecule of claim 13, wherein said antisense region comprises one or more 2'-deoxy-2'-fluoro pyrimidine nucleotides and one or more 2'-O-methyl purine nucleotides.

28. The siNA molecule of claim 13, wherein any pyrimidine nucleotides present in said antisense region comprise 2'-deoxy-2'-fluoro pyrimidine nucleotides and wherein any purine nucleotides present in said antisense region comprise 2'-O-methyl purine nucleotides.

29. The siNA molecule of claim 19, wherein any nucleotides comprising a 3'-terminal nucleotide overhang that are present in said antisense region are 2'-deoxy nucleotides.

30. The siNA molecule of claim 28, wherein said antisense region comprises a phosphorothioate internucleotide linkage at the 3' end of said antisense region.

31. The siNA molecule of claim 13, wherein said antisense region comprises a glyceryl modification at the 3' end of said antisense region.

32. The siNA molecule of claim 19, wherein said 3'-terminal overhangs comprise deoxyribonucleotides .

33. A short interfering nucleic acid (siNA) molecule that down-regulates expression of one or more Myb genes by RNA interference.

34. The siNA molecule of claim 33, wherein the Myb gene is c-Myb.

35. The siNA molecule of claim 33, wherein said siNA molecule comprises no ribonucleotides.

36. The siNA molecule of claim 33, wherein said siNA molecule comprises ribonucleotides.

37. The siNA molecule of claim 33, wherein said siNA molecule is double stranded.

38. The siNA molecule of claim 37, wherein said siNA molecule comprises an antisense strand including nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a Myb protein, and wherein said siNA molecule further comprises a sense strand, wherein said sense strand comprises nucleotide sequence corresponding to nucleotide sequence of a Myb gene or a portion thereof.

39. The siNA molecule of claim 38, wherein said antisense strand and said sense strand each comprise about 19 to about 29 nucleotides, and wherein said antisense strand and said sense strand share at least about 19 complementary nucleotides.

40. The siNA molecule of claim 37, wherein said siNA molecule comprises an antisense region including nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a Myb protein, and wherein said siNA molecule further comprises a sense region, wherein said sense region comprises nucleotide sequence corresponding to nucleotide sequence of Myb gene or a portion thereof.

41. The siNA molecule of claim 40, wherein said antisense region and said sense region each comprise about 19 to about 29 nucleotides, and wherein said antisense region and said sense region share at least about 19 complementary nucleotides.

42. The siNA molecule of claim 33, wherein said siNA molecule is single stranded.

43. The siNA molecule of claim 42, wherein said siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a Myb protein.

44. The siNA molecule of claim 43, wherein said siNA molecule comprises a sequence having about 19 to about 29 nucleotides.

45. The siNA molecule of claim 33, wherein said siNA molecule comprises a sense region and an antisense region and wherein said antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a Myb protein and said sense region comprises sequence complementary to said antisense region.

46. The siNA molecule of claim 33, wherein said siNA molecule is assembled from two oligonucleotide fragments, wherein one oligonucleotide fragment comprises a sense region and a second oligonucleotide fragment comprises an antisense region of said siNA molecule.

47. The siNA molecule of claim 45, wherein said sense region and said antisense region comprise separate oligonucleotides.

48. The siNA molecule of claim 45, wherein said sense region and said antisense region are connected via a linker molecule.

49. The siNA molecule of claim 48, wherein said linker molecule is a polynucleotide linker.

50. The siNA molecule of claim 48, wherein said linker molecule is a non-nucleotide linker.

51. The siNA molecule of claim 45, wherein said sense region comprises a 3'-terminal overhang and said antisense region comprises a 3 '-terminal overhang.

52. The siNA molecule of claim 51, wherein said 3'-terminal overhangs each comprise about 2 nucleotides.

53. The siNA molecule of claim 51, wherein said antisense region 3'-terminal overhang is complementary to RNA encoding a Myb protein.

54. The siNA molecule of claim 45, wherein said sense region comprises one or more 2'-O-methyl pyrimidine nucleotides and one or more 2'-deoxy purine nucleotides.

55. The siNA molecule of claim 45, wherein any pyrimidine nucleotides present in said sense region comprise 2'-deoxy-2'-fluoro pyrimidine nucleotides and wherein any purine nucleotides present in said sense region comprise 2'-deoxy purine nucleotides.

56. The siNA molecule of claim 51, wherein any nucleotides comprising said 3'-terminal nucleotide overhang that are present in said sense region are 2'-deoxy nucleotides.

57. The siNA molecule of claim 45, wherein said sense region comprises a 3'-end and a

5'-end, and wherein a terminal cap moiety is present at the 5'-end, the 3'-end, or both of the 5' and 3' ends of said sense region.

58. The siNA molecule of claim 57, wherein said terminal cap moiety is an inverted deoxy abasic moiety.

59. The siNA molecule of claim 45, wherein said antisense region comprises one or more 2'-deoxy-2'-fluoro pyrimidine nucleotides and one or more 2'-O-methyl purine nucleotides.

60. The siNA molecule of claim 45, wherein any pyrimidine nucleotides present in said antisense region comprise 2'-deoxy-2'-fluoro pyrimidine nucleotides and wherein any purine nucleotides present in said antisense region comprise 2'-O-methyl purine nucleotides.

61. The siNA molecule of claim 51, wherein any nucleotides comprising a 3 '-terminal nucleotide overhang that are present in said antisense region are 2'-deoxy nucleotides.

62. The siNA molecule of claim 60, wherein said antisense region comprises a phosphorothioate internucleotide linkage at the 3' end of said antisense region.

63. The siNA molecule of claim 45, wherein said antisense region comprises a glyceryl modification at the 3' end of said antisense region.

64. The siNA molecule of claim 51, wherein said 3'-terminal overhangs comprise deoxyribonucleotides.