EP1501853A2 - DURCH RNA-INTERFERENZ VERMITTELTE HEMMUNG DER GENEXPRESSION DES REZEPTORS DES EPIDERMALEN WACHSTUMSFAKTORS UNTER VERWENDUNG VON siNA (SHORT INTERFERING NUCLEIC ACID) - Google Patents

DURCH RNA-INTERFERENZ VERMITTELTE HEMMUNG DER GENEXPRESSION DES REZEPTORS DES EPIDERMALEN WACHSTUMSFAKTORS UNTER VERWENDUNG VON siNA (SHORT INTERFERING NUCLEIC ACID)

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Publication number
EP1501853A2
EP1501853A2 EP03716093A EP03716093A EP1501853A2 EP 1501853 A2 EP1501853 A2 EP 1501853A2 EP 03716093 A EP03716093 A EP 03716093A EP 03716093 A EP03716093 A EP 03716093A EP 1501853 A2 EP1501853 A2 EP 1501853A2
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EP
European Patent Office
Prior art keywords
sina
nucleotides
sina molecule
molecule
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP03716093A
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English (en)
French (fr)
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EP1501853A4 (de
Inventor
James Mcswiggen
Pamela Pavco
Leonid Beigelman
Kathy Fosnaugh
Sharon Jamison
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Sirna Therapeutics Inc
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Sirna Therapeutics Inc
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Publication date
Priority claimed from PCT/US2002/016840 external-priority patent/WO2002097114A2/en
Priority claimed from US10/251,117 external-priority patent/US20030170891A1/en
Priority claimed from US10/277,494 external-priority patent/US20030186909A1/en
Application filed by Sirna Therapeutics Inc filed Critical Sirna Therapeutics Inc
Priority claimed from PCT/US2003/005045 external-priority patent/WO2003070912A2/en
Publication of EP1501853A2 publication Critical patent/EP1501853A2/de
Publication of EP1501853A4 publication Critical patent/EP1501853A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/332—Abasic residue
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases that respond to the modulation of epidermal growth factor receptor (EGFR) gene expression.
  • 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 regulation of epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • 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 epidermal growth factor receptor (EGFR) genes, including HER1, HER2, HER3 and HER4 gene expression.
  • siNA short interfering nucleic acid
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • 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 frdni 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.
  • dsRNAs double-stranded RNAs
  • 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.
  • dsRNAs ribonuclease III enzyme
  • 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).
  • RISC RNA-induced silencing complex
  • 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, 10, 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.
  • 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.
  • PKR double-stranded RNA-dependent protein kinase
  • Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in siRNA molecules.
  • 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.
  • 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.
  • 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.
  • Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.
  • Zernicka-Goetz et al International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain dsRNA molecules.
  • Fire et al International PCT Publication No. WO 99/32619, describe particular methods for introducing certain dsRNA molecules into cells for use in inhibiting gene expression.
  • Plaetinck et al International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific dsRNA molecules.
  • Mello et al International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi.
  • Deschamps Depaillette et al International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents.
  • Waterhouse et al International PCT Publication No. 99/53050, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs.
  • Driscoll et al International PCT Publication No. WO 01/49844, describe specific DNA constructs for use in facilitating gene silencing in targeted organisms.
  • RNAi and gene-silencing systems have reported on various RNAi and gene-silencing systems. For example, Parrish et al, 2000, Molecular Cell, 6, 1977-1087, describe specific chemically-modified siRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al, International PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al, International PCT Publication No. WO 01/53475, describe certain methods for isolating a Neurospora silencing gene and uses thereof.
  • Reed et al International PCT Publication No. WO 01/68836, describe certain methods for gene silencing in plants.
  • Honer et al International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs.
  • Deak et al International PCT Publication No. WO 01/72774, describe certain Drosophila-de ⁇ ved gene products that may be related to RNAi in Drosophila.
  • Arndt et al International PCT Publication No. WO 01/92513, describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al, International PCT Publication No.
  • WO 02/44321 describe certain synthetic siRNA constructs.
  • Pachuk et al, International PCT Publication No. WO 00/63364, and Satishchandran et al, International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain dsRNAs.
  • Echeve ⁇ i ⁇ t al, International PCT Publication No. WO 02/38805 describe certain C. elegans genes identified via RNAi.
  • Kreutzer et al, International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 Bl describes certain methods for inhibiting gene expression using RNAi.
  • nucleic acid molecules including short interfering nucleic acid (siNA) molecules, that target EGFR genes.
  • siNA short interfering nucleic acid
  • This invention relates to compounds, compositions, and methods useful for modulating epidermal growth factor receptor (EGFR) function and/or gene expression in a cell 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 hairpin RNA (shRNA) molecules.
  • siNA short interfering nucleic acid
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • the instant invention small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods to modulate the expression of an epidermal growth factor receptor (EGFR), such as HER1, HER2, HER3 and HER4.
  • EGFR epidermal growth factor receptor
  • 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 mvention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating EGFR gene expression or activity in cells by RNA interference (RNAi).
  • siNA synthetic short interfering nucleic acid
  • RNAi RNA interference
  • 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.
  • the invention features about one or more siNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding epidermal growth factor receptors.
  • the present invention features siNA molecules that modulate the expression of EGFR genes HERl (for example Genbank Accession No. NM_005228), HER2 (erbB2/neu) (for example Genbank Accession No. NM_004448), HER3 (for example Genbank Accession No. NM_001982), and HER4 (for example Genbank Accession No. NM_005235).
  • EGFR epidermal growth receptor
  • HERl epidermal growth receptor
  • HER2 HER3, and HER4
  • EGFR genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, collectively referred to hereinafter as EGFR.
  • the various aspects and embodiments are also directed to other genes that express EGFR proteins and other receptors involved in oncogenesis. Those additional genes can be analyzed for target sites using the methods described for EGFR. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.
  • the invention features a siNA molecule having RNAi activity against a EGFR gene, wherein the siNA molecule comprises nucleotide sequence complementary to nucleotide sequence of a EGFR gene, such as those EGFR sequences having GenBank Accession Nos. shown in Table I.
  • a siNA molecule of the invention comprises nucleotide sequence that can interact with nucleotide sequence of a EGFR gene and thereby mediate silencing of EGFR gene expression, for example, wherein the siNA mediates regulation of EGFR gene expression by cellular processes that modulate the chromatin structure of the EGFR gene and prevent transcription of the EGFR gene.
  • 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 EGFR gene.
  • the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA constract complementary to a sequence or portion of sequence comprising a EGFR gene sequence.
  • the invention features a siNA molecule that down regulates expression of an epidermal growth factor receptor (EGFR) gene by RNA interference.
  • EGFR gene can comprise, for example, HERl sequence, HER2 sequence, HER3 sequence, or HER4 sequence and/or any combination thereof.
  • the invention features a siNA molecule having RNAi activity against EGFR RNA, wherein the siNA molecule comprises a sequence complementary to any RNA having EGFR encoding sequence and/or EGFR RNA, such as those sequences having GenBank Accession Nos. shown in Table I. Chemical modifications as shown in Table VIII or otherwise described herein can be applied to any siNA construct of the invention.
  • siNA molecules can be designed to target a class of EGFR genes (e.g., HERl, HER2, HER3, and/or HER4) or alternately specific EGFR genes by selecting sequences that are either shared amongst different EGFR targets or that are alternately unique for a specific EGFR target (e.g., HERl, HER2, HER3, or HER4). Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of EGFR RNA sequence having homology between several EGFR genes so as to target several epidermal growth factor receptors with one siNA molecule.
  • the siNA molecule can be designed to target a sequence that is unique to a specific EGFR RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.
  • the antisense region of HER2 siNA constmcts can comprise a sequence complementary to sequence having any of SEQ ID NOs. 1-249 or 1113-1116.
  • the antisense region can also comprise sequence having any of SEQ ID NOs. 250-498, 1125-1128, 1133-1136, 1141-1144, 1254, 1256, 1258, 1260, 1262, or 1263.
  • the sense region of HER2 constructs can comprise sequence having any of SEQ ID NOs.
  • the sense region can comprise a sequence of SEQ ID NO. 1242 and the antisense region can comprise a sequence of SEQ ID NO. 1243.
  • the sense region can comprise a sequence of SEQ ID NO. 1244 and the antisense region can comprise a sequence of SEQ ID NO. 1245.
  • the sense region can comprise a sequence of SEQ ID NO. 1246 and the antisense region can comprise a sequence of SEQ ID NO. 1247.
  • the sense region can comprise a sequence of SEQ ID NO. 1248 and the antisense region can comprise a sequence of SEQ ID NO. 1249.
  • the sense region can comprise a sequence of SEQ ID NO. 1250 and the antisense region can comprise a sequence of SEQ ID NO. 1251.
  • the sense region can comprise a sequence of SEQ ID NO. 1248 and the antisense region can comprise a sequence of SEQ ID NO. 1252. .
  • the antisense region of EGFR (HERl) siNA constructs' can comprise a sequence complementary to sequence having any of SEQ ID NOs. 499-805 or 1117-1120.
  • the antisense region can also comprise sequence having any of SEQ ID NOs. 806-1112, 1149-1152, 1157-1160, or 1165-1168.
  • the sense region of EGFR (HERl) constructs can comprise sequence having any of SEQ ID NOs. 499-805, 1117-1120, 1145-1148, 1153-1156, or 1161-1164.
  • the sense region can comprise a sequence of SEQ ID NO. 1242 and the antisense region can comprise a sequence of SEQ ID NO. 1243.
  • the sense region can comprise a sequence of SEQ ID NO. 1244 and the antisense region can comprise a sequence of SEQ ID NO. 1245.
  • the sense region can comprise a sequence of SEQ ID NO. 1246 and the antisense region can comprise a sequence of SEQ ID NO. 1247.
  • the sense region can comprise a sequence of SEQ ED NO. 1248 and the antisense region can comprise a sequence of SEQ ID NO. 1249.
  • the sense region can comprise a sequence of SEQ ID NO. 1250 and the antisense region can comprise a sequence of SEQ ID NO. 1251.
  • the sense region can comprise a sequence of SEQ ID NO. 1248 and the antisense region can comprise a sequence of SEQ TD NO. 1252.
  • a siNA construct of the invention comprises sequence having any of SEQ TD NOs. 1-1200 and 1202-1263. The sequences shown in SEQ ID NOs: 1-
  • a siNA molecule of the invention can comprise any contiguous
  • EGFR sequence e.g., about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) contiguous EGFR nucleotides).
  • the invention features a siNA molecule comprising a sequence, for example the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank
  • 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 EGFR 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.
  • 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 EGFR 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.
  • a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a EGFR protein.
  • the siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a EGFR gene or a portion thereof.
  • a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a EGFR protein.
  • the siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a EGFR gene or a portion thereof.
  • a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a EGFR gene. Because related genes typically share some degree of sequence homology with each other, siNA molecules can be designed to target a class of EGFR genes or alternately specific EGFR genes by selecting sequences that are either shared amongst different EGFR targets or alternatively that are unique for a specific EGFR target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of EGFR RNA sequence having homology between several EGFR genes so as to target several EGFR 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 EGFR RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.
  • nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules
  • 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.
  • 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.
  • the invention features about one or more chemically-modified siNA constructs having specificity for EGFR (e.g., HERl, HER2, HER3, and/or HER4) expressing nucleic acid molecules, such as RNA encoding a EGFR protein.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • Non- limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base” nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation.
  • These chemical modifications when used in various siNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al, supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constracts.
  • 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.
  • a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule.
  • a siNA molecule of the mvention can generally comprise about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%,
  • modified nucleotides 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, the antisense strand, or both the sense and antisense strands.
  • 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.
  • 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 seram.
  • 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.
  • 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.
  • 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 EGFR 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.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • 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:
  • each Rl 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 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 the 3' and 5'-ends of the sense strand, the antisense strand, or both strands.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against EGFR (e.g., HERl, HER2, HER3, and or HER4) 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:
  • siNA short interfering nucleic acid
  • each R3, R4, R5, R6, R7, R8, RIO, Rl 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, polyalklyla
  • 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 nucleotides or non-nucleotides of Formula II at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand, the antisense strand, or both strands.
  • 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.
  • 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.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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:
  • siNA short interfering nucleic acid
  • each R3, R4, R5, R6, R7, R8, R10, Rl 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, polyalklyla
  • the chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands.
  • the siNA molecules of the invention can comprise about one or more chemically-modified nucleotides or non-nucleotides of Formula III at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand, the antisense strand, or both strands.
  • 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 Fo ⁇ nula III at the 5 '-end of the sense strand, the antisense strand, or both strands.
  • 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.
  • 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.
  • 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.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • the chemical modification comprises a 5 '-terminal phosphate group having Formula IN:
  • each X and Y is independently O, S, ⁇ , alkyl, substituted alkyl, or alkylhalo; wherein each Z and W is independently O, S, ⁇ , alkyl, substituted alkyl, O-alkyl, S- alkyl, alkaryl, aralkyl, or alkylhalo; and wherein W, X, Y and Z are not all O.
  • the invention features a si ⁇ A molecule having a 5 '-terminal phosphate group having Formula IN on the target-complementary strand, for example a strand complementary to a target R ⁇ A, wherein the si ⁇ A molecule comprises an all RNA siNA molecule.
  • the invention features a siNA molecule having a 5 '-terminal phosphate group having Formula IN on the target-complementary strand wherein the si ⁇ A 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.
  • a 5'-terminal phosphate group having Formula IV is present on the target-complementary strand of a si ⁇ A molecule of the invention, for example a si ⁇ A molecule having chemical modifications having any of Formulae I-NII.
  • the invention features a chemically-modified short interfering nucleic acid (si ⁇ A) molecule capable of mediating R ⁇ A interference (R ⁇ Ai) against EGFR (e.g., HERl, HER2, HER3, and/or HER4) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises about one or more phosphorothioate internucleotide linkages.
  • si ⁇ A short interfering nucleic acid
  • the invention features a chemically-modified short interfering nucleic acid (si ⁇ A) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one si ⁇ A strand
  • the invention features a chemically-modified short interfering nucleic acid (si ⁇ A) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both si ⁇ A strands.
  • the phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the si ⁇ A duplex, for example in the sense strand, the antisense strand, or both strands.
  • the si ⁇ A 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.
  • an exemplary si ⁇ A 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, h another non-limiting example, an exemplary si ⁇ A 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.
  • an exemplary si ⁇ A 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.
  • 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,
  • 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.
  • 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'-O-methyl, 2'-deoxy-2'-fiuoro, 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'-
  • 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.
  • 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'-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,
  • 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.
  • 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'-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 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)
  • 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.
  • 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.
  • siNA short interfering nucleic acid
  • the invention features a siNA molecule comprising about one or more 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.
  • 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.
  • a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically- modified, wherein each strand is about 18 to about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in length, wherein the duplex has about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII.
  • 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-NII 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
  • a siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification comprising a stracture having any of Formulae I-NII or any combination thereof.
  • an exemplary chemically- modified si ⁇ A 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-NII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 base pairs and a 2-nucleotide 3 '-terminal nucleotide overhang, h another embodiment, a linear hairpin si ⁇ A molecule of the invention contains a stem loop motif, wherein the loop portion of the si ⁇ A molecule is biodegradable.
  • a linear hai ⁇ in si ⁇ A molecule of the invention is designed such that degradation of the loop portion of the si ⁇ A molecule in vivo can generate a double-stranded si ⁇ A molecule with 3 '-terminal overhangs, such as 3 '-terminal nucleotide overhangs comprising about 2 nucleotides.
  • a si ⁇ A molecule of the invention comprises a circular nucleic acid molecule, wherein the si ⁇ A 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 si ⁇ A can include a chemical modification, which comprises a stracture having any of Formulae I-NII or any combination thereof.
  • an exemplary chemically-modified si ⁇ A 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 about 2 loops.
  • a circular si ⁇ A molecule of the invention contains two loop motifs, wherein one or both loop portions of the si ⁇ A molecule is biodegradable.
  • a circular si ⁇ A 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.
  • 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:
  • each R3, R4, R5, R6, R7, R8, RIO, 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,
  • 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:
  • each R3, R4, R5, R6, R7, R8, RIO, 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,
  • a siNA molecule of the invention comprises at least one
  • each Rl, 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, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O- aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkly
  • This modification is refe ⁇ ed to herein as "glyceryl" (for example modification 6 in Figure 10).
  • a moiety having any of Formulae 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.
  • 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.
  • a moiety having Formula VII can be present at the 3'-end or the 5'-end of a hai ⁇ in siNA molecule as described herein.
  • a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Fo ⁇ nula 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.
  • 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.
  • LNA locked nucleic acid
  • 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.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2'- deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2'- deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2'-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine nucleotides or alternate
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fiuoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2'-O-mefhyl purine nucleotides (e.g., wherein all purine nucleotides are 2 '-O-methyl purine
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the antisense region are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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 nucleotides), and where about one or more purine nucleotides present in the sense region are 2'-deoxy purine nucleotides (e.g., wherein
  • the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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 nucleo
  • the mvention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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 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 nucleot
  • 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.
  • 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).
  • 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.
  • LNA locked nucleic acid
  • MOE methoxyethoxy
  • the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against EGFR (e.g., HERl, HER2, HER3, and/or HER4) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule.
  • the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker.
  • 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.
  • the conjugate molecule is attached at the 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3'-end and 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof, hi 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, hi 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 , inco ⁇ orated by reference herein.
  • the type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity.
  • 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.
  • 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.
  • 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.
  • the nucleotide linker can be a nucleic acid aptamer.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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, 15:6353 and Nucleic Acids Res. 1987, 75:3113; Cload and Schepartz, J Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 713:5109; Ma et al, Nucleic Acids Res.
  • non-nucleotide further means any group or compound that can be inco ⁇ orated 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.
  • the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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.
  • siNA RNA interference
  • a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA comprise separate oligonucleotides not having any ribonucleotides (e.g., nucleotides having a 2'-OH group) present in the oligonucleotides.
  • the sense and antisense regions of the siNA comprise separate oligonucleotides not having any ribonucleotides (e.g., nucleotides having a 2'-OH group) present in the oligonucleotides.
  • a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as desrcibed herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2'-OH group) present in the oligonucleotide.
  • ribonucleotides e.g., nucleotides having a 2'-OH group
  • 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.
  • a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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.
  • the single stranded siNA molecule of the invention comprises a 5 '-terminal phosphate group.
  • 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. In yet another embodiment, the single stranded siNA molecule of the invention comprises about one or more chemically modified nucleotides or non-nucleotides described herein.
  • 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.
  • a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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'-O-methyl purine nucle
  • a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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
  • a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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 (L
  • a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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 '-methoxy
  • 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.
  • 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).
  • 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.
  • the invention features a method for modulating the expression of a EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the EGFR gene in the cell.
  • a EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method for modulating the expression of a EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR 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 EGFR gene in the cell.
  • 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 EGFR gene and wherein the sense strand sequence of the siNA comprises a sequence identical to the sequence of the target RNA.
  • the invention features a method for modulating the expression of more than one EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the EGFR genes in the cell.
  • EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method for modulating the expression of more than one EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR 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 EGFR genes in the cell.
  • 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 EGFR gene and wherein the sense strand sequence of the siNA comprises a sequence identical to the sequence of the target RNA.
  • siNA molecules of the invention are used as reagents in ex vivo applications.
  • siNA reagents are intoduced into tissue or cells that are transplanted into a subject for therapeutic effect.
  • the cells and/or tissue can be derived from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation.
  • the siNA molecules can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo, hi one embodiment, certain target cells from a patient are extracted.
  • These extracted cells are contacted with siNAs targeteing a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells (e.g. using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells).
  • delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells.
  • the cells are then reintroduced back into the same patient or other patients.
  • the invention features a method of modulating the expression of a EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR 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 EGFR gene in the tissue explant.
  • a EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • 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 EGFR gene in that organism.
  • the invention features a method of modulating the expression of a EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR 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 EGFR gene in the tissue explant.
  • 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 modul
  • the invention features a method of modulating the expression of more than one EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR 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 EGFR genes in the tissue explant.
  • 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 EGFR genes in that organism.
  • the invention features a method of modulating the expression of a EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the EGFR gene in the organism.
  • a EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method of modulating the expression of more than one EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR genes; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the EGFR genes in the organism.
  • EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method for modulating the expression of a EGFR gene (e.g., HERl, HER2, HER3, and or HER4) 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 EGFR gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the EGFR gene in the cell.
  • a EGFR gene e.g., HERl, HER2, HER3, and or HER4
  • the invention features a method for modulating the expression of more than one EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR gene; and (b) contacting the siNA molecule with a cell in vitro or in vivo under conditions suitable to modulate the expression of the EGFR genes in the cell.
  • EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method of modulating the expression of a EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR 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 EGFR gene in the tissue explant.
  • 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 EGFR gene in that organism.
  • the invention features a method of modulating the expression of more than one EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR 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 EGFR genes in the tissue explant.
  • 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 EGFR genes in that organism.
  • the invention features a method of modulating the expression of a EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the EGFR gene in the organism.
  • a EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method of modulating the expression of more than one EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR gene; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the EGFR genes in the organism.
  • EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method of modulating the expression of a EGFR gene (e.g., HERl, HER2, HER3, and or HER4) in an organism comprising contacting the organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the EGFR gene in the organism.
  • a EGFR gene e.g., HERl, HER2, HER3, and or HER4
  • the invention features a method of modulating the expression of more than one EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR genes in the organism.
  • EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • the siNA molecules of the invention can be designed to inhibit target (EGFR) gene expression through RNAi targeting of a variety of RNA molecules.
  • the siNA molecules of the invention are used to target various RNAs co ⁇ esponding to a target gene.
  • 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.
  • 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 polymo ⁇ hism 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).
  • the siNA molecules of the invention are used to target conserved sequences co ⁇ esponding to a gene family or gene families such as EGFR genes (e.g., HERl, HER2, HER3, and HER4).
  • EGFR genes e.g., HERl, HER2, HER3, and HER4.
  • siNA molecules targeting multiple EGFR targets can provide increased therapeutic effect.
  • siNA can be used to characterize pathways of gene function in a variety of applications.
  • 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 cancer or other proliferative diseases and disorders.
  • siNA molecule(s) and/or methods of the invention are used to inhibit the expression of gene(s) that encode RNA refe ⁇ ed to by Genbank Accession, for example EGFR genes (e.g., HERl, HER2, HER3, and/or HER4) encoding RNA sequence(s) refe ⁇ ed to herein by Genbank Accession number, for example Genbank Accession Nos. shown in Table I.
  • Genbank Accession for example EGFR genes (e.g., HERl, HER2, HER3, and/or HER4) encoding RNA sequence(s) refe ⁇ ed to herein by Genbank Accession number, for example Genbank Accession Nos. shown in Table I.
  • the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites witlrin the target RNA sequence.
  • the siNA molecules of (a) have strands of a fixed length, for example, about 23 nucleotides in length.
  • 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, h one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein.
  • the assay can comprise a cell culture system in which target RNA is expressed, hi another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence.
  • the target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.
  • 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 construct strands (e.g., for a siNA constract having 21 nucleotide sense and antisense strands with
  • the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length.
  • 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.
  • fragments of EGFR 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 EGFR RNA sequence.
  • the target EGFR 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.
  • 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, h 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.
  • the assay can comprise a reconstituted in vitro siNA assay as described herein.
  • 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.
  • 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.
  • detecttable 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 about 1 to about 5% of the target RNA is sufficient to detect above the background for most methods of detection.
  • the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent.
  • 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 earner or diluent,
  • 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, hi yet another embodiment, the invention features a method for reducing or preventing tissue rejection in a subject comprising administering to the subject a composition of the invention under conditions suitable for the reduction or prevention of tissue rej ection in the subj ect.
  • the invention features a method for validating a EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR target gene; (b) introducing the siNA molecule into a cell, tissue, or organism under conditions suitable for modulating expression of the EGFR 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.
  • a EGFR gene e.g., HERl, HER2, HER3, and/or HER4 target
  • 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 EGFR target gene
  • the invention features a method for validating a EGFR gene (e.g., HERl, HER2, HER3, and/or HER4) 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 EGFR target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the EGFR target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.
  • a EGFR gene e.g., HERl, HER2, HER3, and/or HER4 target
  • 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 EGFR target gene
  • biological system is meant, material, in a purified or unpurified fonn, 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.
  • biological system includes, for example, a cell, tissue, or organism, or extract thereof.
  • biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.
  • 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).
  • 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.
  • GFP Green Florescent Protein
  • 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 EGFR target gene in a cell, tissue, or organism, h 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 EGFR target gene in a cell, tissue, or organism.
  • the invention features a cell containing about one or more siNA molecules of the invention, which can be chemically-modified.
  • the cell containing a siNA molecule of the invention is a mammalian cell.
  • the cell containing a siNA molecule of the invention is a human cell.
  • the synthesis of a siNA molecule of the invention 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, hi another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis, hi yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.
  • the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second
  • 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.
  • 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.
  • CPG controlled pore glass
  • a cleavable linker such as a succinyl linker
  • 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.
  • 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.
  • the chemical moiety, such as a dimethoxytrityl group is removed during purification, for example, using acidic conditions.
  • 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.
  • the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for
  • 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.
  • 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.
  • the invention features a method for making a doublestranded 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.
  • 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, inco ⁇ orated by reference herein in their entirety.
  • the invention features siNA constracts that mediate RNAi against EGFR (e.g., HERl, HER2, HER3, and/or HER4), wherein the siNA construct comprises about one or more chemical modifications, for example, about one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA constract.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • the siNA construct comprises about one or more chemical modifications, for example, about one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA constract.
  • 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.
  • the invention features siNA constracts that mediate RNAi against EGFR (e.g., HERl, HER2, HER3, and/or HER4), 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.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • 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.
  • the invention features siNA constracts that mediate RNAi against EGFR (e.g., HERl, HER2, HER3, and/or HER4), wherein the siNA construct comprises about one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA constract and a complementary target RNA sequence within a cell.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • the invention features siNA constracts that mediate RNAi against EGFR (e.g., HERl, HER2, HER3, and/or HER4), 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 constract and a complementary target DNA sequence within a cell.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • 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) infroducing 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 RNA sequence.
  • the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence comprising (a) introducing nucleotides having any of 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 DNA sequence.
  • the invention features siNA constructs that mediate RNAi against EGFR (e.g., HERl, HER2, HER3, and/or HER4), 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.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • 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.
  • the invention features chemically-modified siNA constructs that mediate RNAi against EGFR (e.g., HERl, HER2, HER3, and/or HER4) 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 constructs.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method for generating siNA molecules with improved RNAi activity against EGFR (e.g., HERl, HER2, HER3, and/or HER4), 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.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method for generating siNA molecules with improved RNAi activity against a EGFR target RNA (e.g., HERl , HER2, HER3, and/or HER4) 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.
  • a target RNA e.g., HERl , HER2, HER3, and/or HER4
  • the invention features a method for generating siNA molecules with improved RNAi activity against a EGFR target DNA (e.g., HERl, HER2, HER3, and/or HER4) 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.
  • a target DNA e.g., HERl, HER2, HER3, and/or HER4
  • the invention features siNA constracts that mediate RNAi against EGFR (e.g., HERl, HER2, HER3, and/or HER4), wherein the siNA construct comprises about one or more chemical modifications described herein that modulates the cellular uptake of the siNA constract.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • the invention features a method for generating siNA molecules against EGFR (e.g., HERl, HER2, HER3, and/or HER4) with improved cellular uptake, 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 cellular uptake.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • the invention features siNA constructs that mediate RNAi against EGFR (e.g., HERl, HER2, HER3, and/or HER4), 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.
  • polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct
  • conjugates that target specific tissue types or cell types in vivo are described in Vargeese et al, U.S. Serial No. 10/201,394, inco ⁇ orated by reference herein.
  • the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) infroducing a conjugate into the stracture 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.
  • 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.
  • the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing an excipient fo ⁇ nulation to a siNA molecule; and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.
  • excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, and others.
  • 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.
  • polyethylene glycol 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.
  • prefened components of the kit include the a siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, US 6,395,713).
  • the kit can be used for target validation, such as in determining gene function and/or activity, or in drag optimization, and in drug discovery (see for example Usman et al., USSN 60/402,996).
  • Such a kit can also include instructions to allow a user of the kit to practice the invention.
  • short interfering nucleic acid refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference "RNAi” or gene silencing in a sequence-specific manner; see for example Bass, 2001, Nature, 411, 428-429; Elbashir et al, 2001, Nature, 411, 494-498; and Kreutzer et al, hitemational PCT Publication No.
  • Non limiting examples of siNA molecules of the invention are shown in Figures 4-6, and Tables II, III, and IV herein.
  • 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 conesponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e.
  • each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs); the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence conesponding to the target nucleic acid sequence or a portion thereof.
  • 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 hai ⁇ in 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 conesponding 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 conesponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi.
  • the siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence co ⁇ esponding 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.
  • 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 '-di
  • the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic intercations, and/or stacking interactions.
  • the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene, hi 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.
  • siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
  • 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).
  • siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
  • 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 refe ⁇ ed to as short interfering modified oligonucleotides "siMON.”
  • siNA is meant to be equivalent to other tenns 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 hai ⁇ in RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others, hi 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.
  • 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).
  • modulate is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • modulate can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
  • inhibitor By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention.
  • inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule
  • inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches.
  • inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.
  • RNA nucleic acid that encodes an RNA
  • 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 viras, which is present in the cell after infection thereof.
  • the cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, viras, bacterium, or fungus.
  • Non-limiting examples of plants include monocots, dicots, or gymnosperms.
  • Non-limiting examples of animals include vertebrates or invertebrates.
  • fungi m clude molds or yeasts.
  • sense region is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule, hi addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.
  • antisense region is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence.
  • the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule.
  • 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.
  • EGFR epidermal growth factor receptor protein or polynucleotides encoding an epidermal growth factor receptor protein, such as HERl (e.g., encoded by Genbank Accession No. NM_005228), HER2 (e.g., encoded by
  • Genbank Accession No. NM_004448 Genbank Accession No. NM_004448
  • HER3 e.g., encoded by Genbank Accession No.
  • NM_001982 e.g., encoded by Genbank Accession No. NM_005235.
  • HER4 e.g., encoded by Genbank Accession No. NM_005235.
  • EGFR proteins any epidermal growth factor receptor protein, peptide, polypeptide, or a mutant protein derivative thereof, having epidermal growth factor receptor activity, for example, having the ability to bind an epidermal growth factor and/or having tyrosine kinase activity.
  • nucleotide sequence of about 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.
  • 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.
  • 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.
  • 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., about 5, 6, 7, 8, 9, or 10 out of 10, being about 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 pathologic indications, such as cancer, including but not limited to breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and any other diseases or conditions that are related to or will respond to the levels of EGFR in a cell or tissue, alone or in combination with other therapies.
  • the reduction of EGFR expression specifically EGFR gene RNA levels
  • reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.
  • 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.
  • 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).
  • siNA molecules of the invention comprising hai ⁇ in 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 II- VII.
  • Exemplary synthetic siNA molecules of the invention are shown in Tables IV- VII and/or Figures 4-6. Descriptions of various modified siNA constructs of the invention are described in Table VIII.
  • 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 inco ⁇ oration in biopolymers.
  • the nucleic acid molecules of the invention comprise sequences shown in Tables II- VII and/or Figures 4 and 5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures.
  • the chemically modified constracts described herein can be applied to any siNA sequence of the invention.
  • the invention provides mammalian cells containing about one or more siNA molecules of this invention.
  • the siNA molecules can independently be targeted to the same or different sites.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -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, and 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 refe ⁇ ed to as analogs or analogs of naturally-occurring RNA.
  • 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. In another embodiment, a subject is a human or human cells.
  • phosphorothioate 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.
  • universal base 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-nitropy ⁇ ole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).
  • acyclic nucleotide 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 mvention can be used to treat diseases or conditions discussed herein (e.g., cancer).
  • diseases or conditions discussed herein e.g., cancer
  • 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.
  • the siNA molecules can be used in combination with other known treatments to treat conditions or diseases discussed above.
  • 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.
  • the invention features 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 siNA molecule.
  • 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.
  • the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
  • the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule refened to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in Table I.
  • an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.
  • siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules 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, refroviras, adenovirus, or alphaviras.
  • 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.
  • siNA molecules 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.
  • RNAi RNA interference
  • vectors any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • 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 perfo ⁇ ned such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide.
  • the two siNA sfrands 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 conespond 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
  • RdRP RNA-dependent RNA polymerase
  • siNA duplexes RNA-dependent RNA polymerase
  • 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.
  • RdRP RNA-dependent RNA polymerase
  • Figure 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention, hi 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).
  • N N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N).
  • Various modifications are shown for the sense and antisense strands of the siNA constracts.
  • 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 tenninal 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'- luoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • the sense strand comprises 21 nucleotides wherein the two tenninal 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.
  • 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 tenninal 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.
  • 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 tenninal 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.
  • 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'-fluoro modified nucleotides and all purine nucleotides that may be present are 2'-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • the sense strand comprises 21 nucleotides having 5'- and 3'- terminal cap moieties wherein the two terminal 3 '-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • the antisense strand comprises 21 nucleotides, optionally having a 3 '-terminal glyceryl moiety and wherein the two terminal 3 '-nucleotides are optionally complementary to the target RNA sequence, and having one 3 '-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be present are 2'-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.
  • the antisense strand of constructs A-F comprises 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 an EGFR (HER2) siNA sequence.
  • Figure 6 shows non-limiting examples of different siNA constructs of the invention.
  • the examples shown (constructs 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.
  • Constract 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker.
  • the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of constract 1 in vivo and/or in vitro.
  • constract 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA constract 1 in vivo and/or in vitro.
  • the stability and/or activity of the siNA constracts 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 hai ⁇ in constracts.
  • Figure 7A A DNA oligomer is synthesized with a 5 '-restriction site (Rl) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined EGFR 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.
  • Rl 5 '-restriction site
  • X loop sequence of defined sequence
  • Figure 7B The synthetic constract is then extended by DNA polymerase to generate a hai ⁇ in structure having self-complementary sequence that will result in a siNA transcript having specificity for a EGFR 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 constract can be designed such that a 3'-te ⁇ ninal nucleotide overhang results from the transcription, for example by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al, 2002, Nature Biotechnology, 29, 505-508.
  • Figure 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constracts.
  • Figure 8A A DNA oligomer is synthesized with a 5 '-restriction (Rl) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined EGFR 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).
  • Rl 5 '-restriction
  • Sense region of siNA region having sequence identical (sense region of siNA) to a predetermined EGFR target sequence
  • 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 hai ⁇ in stracture having self-complementary sequence.
  • FIG. 8C The constract is processed by restriction enzymes specific to Rl and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells.
  • the franscription cassette is designed such that a U6 promoter region flanks each side of the dsDNA, which generates the separate sense and antisense strands of the siNA.
  • Poly T termination sequences can be added to the 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.
  • stabilization chemistries (1- 10) that can be used, for example, to stabilize the 3
  • modified and unmodified backbone chemistries indicated in the figure can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I.
  • 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 constructs of the invention that are nuclease resistance while preserving the ability to mediate RNAi activity.
  • Chemical modifications are introduced into the siNA constract based on educated design parameters (e.g. introducing 2'-mofications, 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).
  • the siNA constract is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay).
  • FIG. 12 shows a non-limiting example of reduction of HER2 protein in SK-BR- 3 cells mediated by siNA targeting HER2 mRNA site 2344.
  • SK-BR-3 cells were transfected with 0.39 - 25 nM siNA (RPI#28266/28267) or the inverted control (RPI#28268/28269) as indicated and cationic lipid (4 ⁇ g/mL).
  • HER2 protein levels were measured 48h post-treatment by ELISA.
  • MTS assay The ratio of HER2 protein over cell density (MTS assay) was detennined for each treatment group and results are reported as normalized HER2 protein after treatment with lipid alone, active siNA or inverted control relative to untreated (UNT) cells. Results are reported as the mean of duplicate samples ⁇ SD.
  • Figure 13 shows a non-limiting example of reduction of HER2 mRNA in SK-BR-
  • SK-BR-3 cells were transfected with 0.39 - 25 nM siNA (RPI#28266/28267) or the inverted control (RPI#28268/28269) as indicated and cationic lipid (4 ⁇ g/mL).
  • HER2 mRNA levels were measured 24h post-treatment by real time RT-PCR. The ratio of HER2 mRNA over 36B4 mRNA was determined for each treatment group and results are reported as normalized HER2 mRNA after treatment with lipid alone, active siNA or inverted control relative to untreated (UNT) cells. Results are reported as the mean of triplicate samples ⁇ SD.
  • Figure 14 shows a non-limiting example of antiproliferative activity of either unmodified (RPI#28266/28267) or chemically-modified (RPI#29991/29990) siNAs targeting HER2 site 2344 in SK-BR-3 cells.
  • SK-BR-3 cells were transfected with 6.25 - 50 nM siNA (RPI#28266/28267 or RPI#29991/29990) or inverted controls (RPI#28268/28269 or RPI#29997/29999) as indicated and cationic lipid (4 ⁇ g/mL) on days one and three.
  • Cell proliferation was determined 96h after treatment with lipid alone, active siNAs or inverted controls relative to untreated (UNT) cells. Results are reported as the mean of triplicate samples ⁇ SD.
  • Figure 15 shows a non-limiting example of reduction of HER2 protein in SK-OV-
  • SK-OV-3 cells were transfected with 0.39 - 25 nM siNA (RPI#28266/28267) or the inverted control (RPI#28268/28269) as indicated and cationic lipid (4 ⁇ g/mL).
  • HER2 protein levels were measured 48h post-treatment by ELISA. The ratio of HER2 protein over cell density (MTS assay) was determined for each treatment group and results are reported as normalized HER2 protein after treatment with lipid alone, active siNA or inverted control relative to untreated (UNT) cells. Results are reported as the mean of duplicate samples ⁇ SD.
  • Figure 16 shows a non-limiting example of reduction of HER2 mRNA in SK-OV-
  • SK-OV-3 cells were transfected with 0.39 - 25 nM siNA (RPI#28266/28267) or the inverted control (RPI#28268/28269) as indicated and cationic lipid (4 ⁇ g/mL).
  • HER2 mRNA levels were measured 24h post-treatment by real time RT-PCR. The ratio of HER2 mRNA over 36B4 mRNA was determined for each treatment group and results are reported as normalized HER2 mRNA after treatment with lipid alone, active siNA or inverted control relative to untreated (UNT) cells. Results are reported as the mean of triplicate samples ⁇ SD.
  • Figure 17 shows a non-limiting example of reduction of HER2 mRNA in SK-OV- 3 cells mediated by chemically-modified siNAs that target HER2 mRNA site 2344.
  • SK- OV-3 cells were transfected with 6.25 or 25 nM unmodified siNA (RPI#28266/28267) or the inverted control (RPI#28268/28269) as well as sets of chemically-modified siNAs as indicated and cationic lipid (4 ⁇ g/mL).
  • a particular modified sense strand (RPI#29991) was mixed with each of four possible antisense strands (RPI#s 29990, 29994, 29995 or 29993) and cells were treated with these four sets.
  • HER2 mRNA levels were measured 24h post-treatment by real time RT-PCR. The ratio of HER2 mRNA over 36B4 mRNA was determined for each treatment group and results are reported as normalized HER2 mRNA after treatment with lipid alone, active siNA or inverted control, and modified sets of siNAs relative to untreated (UNT) cells. Results are reported as the mean of triplicate samples ⁇ SD.
  • Figure 18 shows a non-limiting example of reduction of HER2 mRNA in SK-OV- 3 cells mediated by chemically-modified siNAs that target HER2 mRNA site 2344.
  • SK- OV-3 cells were transfected with 6.25 or 25 nM unmodified siNA (RPI#28266/28267) or the inverted control (RPI#28268/28269) as well as sets of chemically-modified siNAs as indicated and cationic lipid (4 ⁇ g/mL).
  • a particular modified sense strand (RPI#29989) was mixed with each of four possible antisense strands (RPI#s 29990, 29994, 29995 or 29993) and cells were treated with these four sets.
  • HER2 mRNA levels were measured 24h post-treatment by real time RT-PCR. The ratio of HER2 mRNA over 36B4 mRNA was detennined for each treatment group and results are reported as normalized HER2 mRNA after treatment with lipid alone, active siNA or inverted confrol, and modified sets of siNAs relative to untreated (UNT) cells. Results are reported as the mean of triplicate samples ⁇ SD.
  • Figure 19 shows a non-limiting example of reduction of HER2 mRNA in SK-OV- 3 cells mediated by chemically-modified siNAs that target HER2 mRNA site 2344.
  • SK- OV-3 cells were fransfected with 6.25 or 25 nM unmodified siNA (RPI#28266/28267) or the inverted control (RPI#28268/28269) as well as sets of chemically-modified siNAs as indicated and cationic lipid (4 ⁇ g/mL).
  • a particular modified sense strand (RPI#29992) was mixed with each of four possible antisense strands (RPI#s 29990, 29994, 29995 or 29993) and cells were treated with these four sets.
  • HER2 mRNA levels were measured 24h post-treatment by real time RT-PCR. The ratio of HER2 mRNA over 36B4 mRNA was determined for each treatment group and results are reported as normalized HER2 mRNA after treatment with lipid alone, active siNA or inverted control, and modified sets of siNAs relative to untreated (UNT) cells. Results are reported as the mean of triplicate samples ⁇ SD.
  • Figure 20 shows a non-limiting example of reduction of EGFR (HERl) mRNA in A549 cells mediated by chemically-modified siNAs that target EGFR mRNA.
  • A549 cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA.
  • a siNA construct comprising ribonucleotides and 3 '-terminal dithymidine caps
  • siNA constructs were also compared to untreated cells, cells transfected with lipid and scrambled siNA constracts (Scraml and Scram2), and cells transfected with lipid alone (fransfection control).
  • both siNA constructs show significant reduction of EGFR RNA expression.
  • Figure 21 shows a non-limiting example of reduction of HER2 mRNA in A549 cells mediated by RNA-based and chemically-modified siNAs that target HER2 mRNA sites 2344 and 3706.
  • A549 cells were fransfected with 4 ug/ml lipid complexed with 25 nM unmodified siNA with a 3 '-terminal dithymidine cap (RPI#28266/28267) or a conesponding inverted control (RPI#28268/28269) for site 2344 and (RPI#28262/28263) and a conesponding inverted control (RPI 28264/28265) for site 3706.
  • A549 cells were transfected with 4 ug/ml lipid complexed with 25 nM modified siNA (RPI#30442/30443) and a co ⁇ esponding matched control (RPI#30444/30445) for site 2344 and (RPI#30438/30439) and a conesponding matched control (RPI 30440/30441) for site 3706.
  • the modified and unmodified constructs targeting sites 2344 and 3706 all demonstrate significant inhibition of HER2 RNA expression.
  • 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.
  • 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.
  • 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,
  • RNA silencing The conesponding process in plants is commonly refened to as post-transcriptional gene silencing or RNA silencing and is also refened to as quelling in fungi.
  • the process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al, 1999, Trends Genet., 15, 358).
  • Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA.
  • dsRNAs double-stranded RNAs
  • 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 '-ohgoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
  • Dicer The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme refened 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 refe ⁇ ed 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).
  • RISC RNA-induced silencing complex
  • RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin stracture 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, 291, 2215-2218; and Hall et al, 2002, Science, 297, 2232-2237).
  • siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post- transcriptional level.
  • RNAi has been studied in a variety of systems. Fire et al, 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol, 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al, 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al, 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells.
  • nucleic acid Molecules 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.
  • small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery.
  • the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and or RNA structure.
  • Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.
  • Oligonucleotides are synthesized using protocols l ⁇ iown 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.
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol 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 IX outlines the amounts and the contact times of the reagents used in the synthesis cycle.
  • syntheses at the 0.2 ⁇ mol 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.
  • 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 I , 49 mM pyridine, 9% water in THF (PERSEPTIVETM). 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 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 transfened 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: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.
  • 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.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc.
  • synthesizer using a 0.2 ⁇ mol 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 IX outlines the amounts and the contact times of the reagents used in the synthesis cycle.
  • syntheses at the 0.2 ⁇ mol 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.
  • Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
  • 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 (PERSEPTIVETM). 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 hitemational Chemical, hie. 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 R ⁇ A is perfo ⁇ ned using either a two-pot or one-pot protocol.
  • the polymer-bound trityl-on oligoribonucleotide is transfened 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:MeC ⁇ :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 ⁇ L of a solution of 1.5 mL N-methylpynolidinone, 750 ⁇ L 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.
  • the polymer-bound trityl-on oligoribonucleotide is transfened 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 r.t. TEA ⁇ HF (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.
  • the quenched NH4HCO3 solution is loaded onto a C-18 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 NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
  • the average stepwise coupling yields are typically about >98% (Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684).
  • nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al, 1992, Science 256, 9923; Draper et al, International PCT publication No. WO 93/23569; Shabarova et al, 1991, Nucleic Acids Research 19, 4247; Bellon et al, 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al, 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.
  • siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex.
  • the linker can be a polynucleotide linker or a non-nucleotide linker.
  • the tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms.
  • the tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.
  • a siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the RNA molecule.
  • 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 inco ⁇ orated herein by reference) and re-suspended in water.
  • 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 constracted based on, but not limited to, adeno-associated viras, refroviras, adenoviras, 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.
  • nucleic acid molecules with modifications can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al, Intemational Publication No. WO
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'- ⁇ - 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).
  • nuclease resistant groups for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'- ⁇ - 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
  • 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.
  • 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.
  • 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.
  • 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 mythylene bicyclo nucleotide (see for example Wengel et al, International PCT Publication No. WO 00/66604 and WO 99/14226).
  • the invention features conjugates and/or complexes of siNA molecules of the invention.
  • 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.
  • 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.
  • the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers.
  • 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.
  • biodegradable linker refers to a nucleic acid or non- nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense sfrands of a siNA molecule of the invention.
  • the biodegradable linker is designed such that its stability can be modulated for a particular pu ⁇ ose, such as delivery to a particular tissue or cell type.
  • the stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'-modified or base modified nucleotides.
  • the biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage.
  • the biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
  • biodegradable refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
  • biologically active molecule refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system.
  • biologically active siNA molecules either alone or in combination with othe 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.
  • phospholipid refers to a hydrophobic molecule comprising at least one phosphorus group.
  • 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.
  • nucleic acid molecules e.g., siNA molecules
  • delivered exogenously optimally are stable within cells until reverse trascription 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.
  • 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.
  • nucleic acid-based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules).
  • combination therapies e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules.
  • the treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A ohgoadenylate, decoys, and aptamers.
  • ribozymes enzymatic nucleic acid molecules
  • allozymes antisense
  • 2,5-A ohgoadenylate 2,5-A ohgoaden
  • 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 strand, or both siNA strands.
  • cap structure is meant chemical modifications, which have been inco ⁇ orated at either terminus of the oligonucleotide (see, for example, Adamic et al, U.S. Pat. No. 5,998,203, inco ⁇ orated 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.
  • 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; t/zre ⁇ -pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5- dihydroxyp
  • 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; tAreo-pentofuranosyl nucleotide; acyclic 3
  • non-nucleotide any group or compound which can be inco ⁇ orated 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.
  • alkyl refers to a saturated aliphatic hydrocarbon, including straight- chain, branched-chain, and cyclic alkyl groups.
  • the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from about 1 to about 7 carbons, more preferably about 1 to about 4 carbons.
  • alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups.
  • the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from about 1 to about 7 carbons, more preferably about 1 to about 4 carbons.
  • 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.
  • the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from about 1 to about 7 carbons, more preferably about 1 to about 4 carbons.
  • 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 comprises carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.
  • the prefened substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
  • alkylaryl 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 about 1 to about 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, py ⁇ olyl, N-lower alkyl pynolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
  • An "amide” refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen.
  • An “ester” refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
  • 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 refened 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, Intemational PCT Publication No.
  • base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents.
  • the invention features modified siNA molecules, with phosphate backbone modifications comprising about one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, mo ⁇ holino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • phosphate backbone modifications comprising about one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, mo ⁇ holino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • abasic 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.
  • unmodified nucleoside is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1' carbon of ⁇ -D-ribo-furanose.
  • modified nucleoside is meant any nucleotide base which contains a modification in the chemical stracture of an unmodified nucleotide base, sugar and/or phosphate.
  • modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.
  • amino 2'-NH or 2'-O-NH 2 , which can be modified or unmodified.
  • 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 inco ⁇ orated by reference in their entireties.
  • nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penefration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
  • a siNA molecule of the invention can be adapted for use to treat for cancer and other indications that can respond to the level of EGFR in a cell or tissue, alone or in combination with other therapies.
  • a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations.
  • Methods for the delivery of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al, 1999, Mol. Membr.
  • 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 inco ⁇ oration 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 proxeinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722).
  • the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.
  • nucleic acid molecules of the invention can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Corny et al, 1999, Clin. Cancer Res., 5, 2330-2337 and Bany et al, Intemational 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.
  • the invention features a pharmaceutical composition
  • 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.
  • a liposome delivery mechanism standard protocols for formation of liposomes can be followed.
  • compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral adminisfration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art.
  • the present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.
  • systemic adminisfration in vivo systemic abso ⁇ tion or accumulation of drags in the blood stream followed by distribution throughout the entire body.
  • Administration routes that lead to systemic abso ⁇ tion include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, mtrapulmonary 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 drag, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.
  • compositions or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.
  • 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 (JoUiet-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.
  • Pluronic P85 which can enhance entry of drags into the CNS
  • 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)
  • nanoparticles such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).
  • Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al, 1998, J Pharm. Sci., 87, 1308-1315; Tyler et al, 1999, FEBS Lett., 421, 280-284; Pardridge et al, 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-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).
  • 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; Ishiwata et al, Chem. Pharm. Bull. 1995, 43, 1005-1011).
  • liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et ⁇ /.,1995, Biochim. Biophys. Ada, 1238, 86-90).
  • the long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol Chem. 1995, 42, 24864-24870; Choi et al, hitemational PCT Publication No.
  • compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a phannaceutically 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 inco ⁇ orated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of / hydroxybenzoic acid.
  • antioxidants and suspending agents can be used.
  • a phannaceutically effective dose is that dose required to prevent, inhibit the occu ⁇ ence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the phannaceutically effective dose depends on the type of disease, the composition used, the route of adminisfration, the type of mammal being freated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount 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.
  • nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable earners, adjuvants and/or vehicles.
  • parenteral as used herein comprises percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like, hi addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier.
  • One or more nucleic acid molecules of the invention can be present in association with about one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients.
  • compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain 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.
  • 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, com 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 abso ⁇ tion in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • 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.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • 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.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl- methylcellulose, sodium alginate, polyvinylpy ⁇ olidone, 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 monoo
  • 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.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • 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.
  • a dispersing or wetting agent exemplified by those already mentioned above.
  • Additional excipients for example sweetening, flavoring and coloring agents, can also be present.
  • compositions of the invention can also be in the form of oil-in- water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavoring agents.
  • Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3- butanediol.
  • Suitable 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.
  • any bland fixed oil can be employed including synthetic mono-or diglycerides.
  • 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.
  • 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 drag.
  • 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 drag.
  • 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.
  • 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 from about 1 mg to about 500 mg of an active ingredient.
  • 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 adminisfration, and rate of excretion, drag combination and the severity of the particular disease undergoing therapy.
  • 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.
  • the invention provides compositions suitable for administering nucleic acid molecules of the invention to specific cell types.
  • 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).
  • ASGPr asialoglycoprotein receptor
  • 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 ohgosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al, 1982, J Biol Chem., 257, 939-945).
  • Lee and Lee, 1987, Glycoconjugate J., 4, 317-328 obtained this high specificity through the use of N- acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose.
  • siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weinfraub, 1985, Science, 229,
  • 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.
  • R ⁇ A molecules of the present invention can be expressed from franscription units (see for example Couture et al, 1996, TIG., 12, 510) inserted into D ⁇ A or R ⁇ A vectors.
  • the recombinant vectors can be D ⁇ A plasmids or viral vectors.
  • si ⁇ A expressing viral vectors can be constructed based on, but not limited to, adeno-associated viras, refroviras, adenoviras, or alphavirus.
  • pol III based constracts are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. ⁇ os. 5,902,880 and 6,146,886).
  • the recombinant vectors capable of expressing the si ⁇ A molecules can be delivered as described above, and persist in target cells.
  • viral vectors can be used that provide for transient expression of nucleic acid molecules.
  • Such vectors can be repeatedly administered as necessary.
  • the si ⁇ A molecule interacts with the target mR ⁇ A and generates an R ⁇ Ai response.
  • Delivery of si ⁇ A 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).
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one si ⁇ A molecule of the instant invention.
  • the expression vector can encode one or both strands of a si ⁇ A duplex, or a single self-complementary strand that self hybridizes into a si ⁇ A duplex.
  • the nucleic acid sequences encoding the si ⁇ A 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).
  • the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a franscription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule.
  • the vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the sequence encoding the siNA of the invention the group comprising and/or an intron (intervening sequences).
  • ORF open reading frame
  • RNA polymerase I RNA polymerase I
  • RNA polymerase II RNA polymerase II
  • RNA polymerase III RNA polymerase 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.
  • 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.
  • transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al, supra; Couture and Stinchcomb, 1996, supra; Noonberg et al, 1994, Nucleic Acid Res., 22, 2830; Noonberg et al, U.S. Pat. No. 5,624,803; Good et al, 1997, Gene Ther., 4, 45; Beigelman et al, International PCT Publication No. WO 96/18736.
  • siNA transcription units can be inco ⁇ orated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenoviras or adeno-associated viras vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
  • plasmid DNA vectors such as adenoviras or adeno-associated viras vectors
  • viral RNA vectors such as retroviral or alphavirus vectors
  • 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 te ⁇ nination 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.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription te ⁇ nination 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.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription te ⁇ nination 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.
  • 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 sfrand of a siNA molecule, wherein the sequence is operably linked to the 3'-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.
  • the epidermal growth factor receptor is a 170 kDa transmembrane glycoprotein consisting of an extracellular 'ligand' binding domain, a transmembrane region and an intracellular domain with tyrosine kinase activity (Kung et al, 1994).
  • the binding of growth factors to the EGFR results in down regulation of the ligand-receptor complex, autophosphorylation of the receptor and other protein substrates, leading ultimately to DNA synthesis and cell division.
  • the external ligand binding domain is stimulated by EGF and also by TGFa, amphiregulin, and some viral growth factors (Modjtahedi & Dean, 1994).
  • c-erbBl avian erythroblastosis viras oncogene
  • v-erbB avian erythroblastosis viras oncogene
  • the v-erbB gene codes for a truncated product that lacks the extracellular ligand binding domain.
  • the tyrosine kinase domain of the EGFR has been found to have 97% homology to the v-erbB transforming protein (Downward et al, 1984).
  • the amplified genes are frequently rea ⁇ anged and associated with polymo ⁇ hism leading to abnormal protein products (Wong et al, 1994).
  • the rea ⁇ angements that have been characterized usually show deletions of part of the extracellular domain, resulting in the production of an EGFR protein that is smaller in size.
  • Three classes of deletion mutant EGF receptor genes have been identified in glioblastoma tumors.
  • Type I mutants lack the majority of the external domain, including the ligand binding site; type II mutants have a deletion in the domain adjacent to the membrane but can still bind ligands; and type III, mutants, which are the most common of the three and are found in 17%) of glioblastomas, have a deletion of 267 amino acids spanning domains I and II of the EGFR gene.
  • abnormal EGFR expression has also been reported in a number of squamous epidermoid cancers and breast cancers (reviewed in Kung et al, 1994; Modjtahedi & Dean, 1994).
  • evidence also suggests that many patients with tumors that over-express EGFR have a poorer prognosis than those having tumors that do not over-express EGFR (Khazaie et al, 1993). Consequently, therapeutic strategies that can potentially inhibit or reduce the abenant expression of EGFR are of great interest as potential anti-cancer agents.
  • HER2 (also known as EGFR2, neu, erbB2 and c-erbB2) is an oncogene that encodes a 185-kDa transmembrane tyrosine kinase receptor.
  • HER2 is a member of the epidermal growth factor receptor (EGFR) family and shares partial homology with other family members. In normal adult tissues HER2 expression is low. However, HER2 is overexpressed in at least 25-30% of breast cancers (McGuire, H.C. and Greene, M.I. (1989) and ovarian cancers (Berchuck et al. (1990) Semin. Oncol. 16: 148-155).
  • HER2 Overexpression of her-2/neu is associated with poor survival in advanced epithelial ovarian cancer (Cancer Research 50: 4087-4091). Furthermore, overexpression of HER2 in malignant breast tumors has been conelated with increased metastasis, chemoresistance and poor survival rates (Slamon et ah, 1987 Science 235: 177-182). Because HER2 expression is high in aggressive human breast and ovarian cancers, but low in normal adult tissues, it is an attractive target for nucleic acid-mediated therapy.
  • EGFR EGFR
  • small interfering nucleic acid (siNA) mediated RNAi a novel approach to the treatment, diagnosis, and study of diseases and conditions related to EGFR (e.g., HERl, HER2, HER3, and/or HER4) activity and/or gene expression.
  • 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.
  • a cleavable linker for example a succinyl-based linker.
  • the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5 -O-DMT group while the complementary strand comprises a terminal 5'-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group.
  • 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 C 18 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.
  • linker coupling conditions includes a hindered base such as diisopropylethylamine (DIP A) and/or DMAP in the presence of an activator reagent such as Bromotripy ⁇ olidinophosphoniumhexaflurorophosphate (PyBrOP).
  • DIP A diisopropylethylamine
  • PyBrOP Bromotripy ⁇ olidinophosphoniumhexaflurorophosphate
  • standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5'-O-DMT intact.
  • 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.
  • siNA duplex 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 NaCl).
  • CV column volume
  • ACN Aqueous with 50mM NaOAc and 50mM NaCl
  • the column is then washed, for example with 1 CV H2O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes.
  • TFA trifluoroacetic acid
  • the remaining TFA solution is removed and the column washed with H20 followed by 1 CV IM NaCl 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 construct in which each peak conesponds to the calculated mass of an individual siNA sfrand of the siNA duplex.
  • the same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably co ⁇ esponding to the duplex siNA, and two peaks presumably conesponding to the separate siNA sequence strands.
  • CGE capillary gel electrophoresis
  • Ion exchange HPLC analysis of the same siNA contract only shows a single peak.
  • RNA target of interest such as a viral or human mRNA transcript
  • sequence of a gene or RNA gene transcript derived from a database is used to generate siNA targets having complementarity to the target.
  • a database such as Genbank
  • 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.
  • RNA franscript 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 about 1 to about 1000 target sites are chosen within the franscript based on the size of the siNA contract 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.
  • the target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example about 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, Mac Vector, or the GCG
  • the siNAs conespond to more than one target sequence such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog.
  • 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.
  • the ranking can indentify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.
  • 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.
  • 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.
  • the ranked siNA subsequences can be further analyzed and ranked according to GC content.
  • a preference can be given to sites containing about 30 to about 70% GC, with a further preference to sites containing about 40 to about 60% GC.
  • the ranked siNA subsequences can be further analyzed and ranked according to self- folding and internal hai ⁇ ins. Weaker internal folds are prefened; strong hai ⁇ in structures are to be avoided.
  • 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 sfrand 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 sfrand because that will place GGG in the antisense strand. 7.
  • the ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3 '-end of the sequence, and/or AA on the 5'-end of the sequence (to yield 3' UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.
  • UU uridine dinucleotide
  • target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) sfrand of the siNA duplex (see Tables II and III). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3' terminal nucleotides of both the sense and antisense sfrands are replaced by TT prior to synthesizing the oligos.
  • siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most prefened target site within the target RNA sequence.
  • a pool of siNA constracts specific to an EGFR (e.g., HERl, HER2) target sequence is used to screen for target sites in cells expressing EGFR RNA (e.g., SK-OV-3 or SK-BR-3 cells).
  • EGFR RNA e.g., SK-OV-3 or SK-BR-3 cells.
  • the general sfrategy 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. 1-249, 1113-1116, 1121- 1124, 1129-1132, 1137-1140, 499-805, 1117-1120, 1145-1148, 1153-1156, and 1161- 1164 and antisense sequences comprising SEQ ID NOs.
  • SK- OV-3/SK-BR-3 cells expressing EGFR are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with EGFR 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 demonstrating a positive phenotypic change e.g., decreased proliferation, decreased EGFR mRNA levels or decreased EGFR protein expression, are sequenced to detennine the most suitable target site(s) within the target EGFR RNA sequence.
  • siNA target sites were chosen by analyzing sequences of the EGFR RNA target (e.g., HERl, HER2, HER3 and/or HER4) and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein.
  • siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity.
  • siNA molecules can be designed to target sites within any known RNA sequence, for example, those RNA sequences conesponding to the any gene franscript.
  • Chemically modified siNA constucts are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an approprate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity.
  • an approprate 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 constracts 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.
  • siNA constracts can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al, US Patent Nos.
  • 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).
  • exocyclic amine protecting groups e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine.
  • 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, inco ⁇ orated by reference herein in its entirety).
  • 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.
  • a capping reagent such as acetic anhydride to yield inactive 5 '-acetyl moieties.
  • the trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage.
  • 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 perfonned as is generally described in Usman et al., US 5,831,071, US 6,353,098, US 6,437,117, Bellon et al., US 6,054,576, US 6,162,909, US 6,303,773, and Scaringe supra, all of which are inco ⁇ orated by reference herein in their entireties.
  • deprotection conditions can be modified to provide the best possible yield and purity of siNA constracts.
  • oligonucleotides comprising 2 '-deoxy-2 '-fluoro nucleotides can degrade under inapproprate deprotection conditions.
  • Such oligonucleotides are deprotected using aqueous methylamine at about 35°C for 30 minutes.
  • 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
  • RNAi in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constracts targeting EGFR 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 EGFR 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 EGFR expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein.
  • Sense and antisense siNA sfrands 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 bv gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide.
  • buffer such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate
  • the Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated.
  • the assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 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 confrol reactions in which siNA is omitted from the reaction.
  • internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha- 32 p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification.
  • target RNA is 5'-32p-end labeled using T4 polynucleotide kinase enzyme.
  • Assays are perfonned 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 hnager® quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.
  • this assay is used to determine target sites the EGFR RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the EGFR RNA target, for example, by analysing the assay reaction by elecfrophoresis of labelled target RNA, or by northern blotting, as well as by other methodology well known in the art.
  • Example 7 Models useful in evaluation nucleic acid inhibition of EGFR siNA molecules targeted to the human EGFR (e.g., HERl, HER2, HER3, and/or
  • HER4 RNA are designed and svnthesi ⁇ ri « A ⁇ ⁇ above. These nucleic acid molecules can be tested for cleavage activity in vitro and in vivo, for example, using the following procedure.
  • the target sequences and the nucleotide location within the EGFR RNA are given in Tables herein.
  • Nucleic acid molecules targeted to the human EGFR RNA are designed and synthesized as described above.
  • a variety of endpoints have been used in cell culture models to evaluate EGFR-mediated effects after treatment with anti-EGFR agents.
  • Phenotypic endpoints include inhibition of cell proliferation, apoptosis assays and reduction of EGFR protein expression. Because overexpression of EGFR is directly associated with increased proliferation of tumor cells, a proliferation endpoint for cell culture assays is preferably used as a primary screen. There are several methods by which this endpoint can be measured.
  • cells are allowed to grow (typically 5 days) after which either the cell viability, the inco ⁇ oration of [3H] thymidine into cellular DNA and/or the cell density can be measured.
  • the assay of cell density is well-known to those skilled in the art and can, for example, be performed in a 96-well format using commercially available fluorescent nucleic acid stains (such as Syto 13 or CyQuant) or the ability of live cells to reduce MTS to formazon (Promega, Madison, WI).
  • fluorescent nucleic acid stains such as Syto 13 or CyQuant
  • MTS assay is described herein.
  • a nucleic acid-mediated decrease in the level of EGFR RNA and/or EGFR protein expression can be evaluated using methods known in the art, such as RT-PCR, Northern blot, ELISA, Western blot, and immunoprecipitation analyses, to name a few techniques.
  • SKBR-3 and SKOV-3 Two human cell lines (SKBR-3 and SKOV-3) that are known to express medium to high levels of EGFR (HERl and HER2) protein are considered for nucleic acid screening, hi order to validate these cell lines for EGFR-mediated sensitivity, both cell lines are freated with an EGFR specific antibody, for example, lnAB IMC-C225 (hnClone) and its effect on cell proliferation is determined.
  • mAB is added to cells at concentrations ranging from 0-8 ⁇ M in medium containing either no seram (OptiMem), 0.1%) or 0.5% FBS and efficacy is determined via cell proliferation.
  • lipids as described in PCT application WO99/05094 lipids as described in PCT application WO99/05094
  • lipids as described in PCT application WO99/05094 lipids as described in PCT application WO99/05094
  • additional description of useful lipids is provided above, and those skilled in the art are also familiar with a variety of lipids that can be used for delivery of oligonucleotide to cells in culture.
  • this panel of lipid delivery vehicles is screened in SKBR-3 and SKOV-3 cells using previously established control oligonucleotides.
  • Specific lipids and conditions for optimal delivery are selected for each cell line based on these screens. These conditions are used to deliver EGFR specific nucleic acids to cells for primary (inhibition of cell proliferation) and secondary (decrease in EGFR RNA/protein) efficacy endpoints.
  • Nucleic acid screens were performed using an automated, high throughput 96-well cell proliferation assay. Cell proliferation was measured over a 5-day treatment period using the MTS assay for determining cell density. The growth of cells treated with siNA/lipid complexes was compared to untreated cells, lipid treatment alone, and to cells treated with a inverted confrol sequence. Inverted controls can no longer bind to the target site due to a reversal of the native sequence. These controls are used to determine non-specific inhibition of cell growth caused by nucleic acid chemistry. The growth of cells treated with siNA/lipid complexes was compared to untreated cells, lipid treatment alone, and to cells treated with an inverted confrol sequence.
  • Lead nucleic acids were chosen from the primary screen based on their ability to inhibit cell proliferation in a specific manner. Dose response assays were canied out on these leads and a subset was advanced into a secondary screen using a reduction in the level of EGFR protein and/or RNA as an endpoint. Secondary Screen: Decrease in EGFR Protein and/or RNA
  • a secondary screen that measures the effect of anti-EGFR nucleic acids on EGFR (e.g., HERl, HER2, HER3, and/or HER4) protein and/or RNA levels is used to affirm preliminary findings.
  • a EGFR ELISA for both SKBR-3 and SKOV-3 cells has been established and made available for use as an additional endpoint.
  • a real time RT-PCR assay (TaqMan assay) has been developed to assess EGFR RNA reduction.
  • Dose response activity of nucleic acid molecules of the instant invention can be used to assess both EGFR protein and RNA reduction endpoints.
  • siNA Mechanism Assays A TaqMan® assay for measuring the siNA-mediated decrease in EGFR (e.g.,
  • RNA has been established. This assay is based on PCR technology and can measure in real time the production of EGFR mRNA relative to a standard cellular mRNA such as 36B4. This RNA assay is used to establish proof that lead siNAs are working through an RNA cleavage mechanism and result in a decrease in the level of EGFR mRNA, thus leading to a decrease in cell surface EGFR protein receptors and a subsequent decrease in tumor cell proliferation.
  • EGFR e.g., HERl, HER2, HER3, and/or HER4
  • nude mice bearing human vulvar (A431), lung (A549 and SK-LC-16 NSCL and LX-1) and prostate (PC-3 and TSU-PRI) xenografts were sensitive to the anti- HER2 tyrosine kinase inhibitor ZD1839 (Iressa), resulting in a partial regression of A431 tumor growth, 70-80% inhibition of tumor growth (A549, SKLC-16, TSU-PRI and PC-3 tumors), and 50-55% inhibition against the LX-1 tumor at a 150 mg kg dose (infraperitoneal, every 3-4 days x 4), (Sirotnak et al, 2000, Clin. Cancer Res., 6, 4885-48892).
  • Tumor cell lines (SKBR-3 and SKOV-3) are characterized to establish their growth curves in mice. These cell lines are implanted into both nude and SCID mice and primary tumor volumes are measured 3 times per week. Growth characteristics of these tumor lines using a Matrigel implantation- fo ⁇ nat can also be established. The use of other cell lines that have been engineered to express high levels of EGFR can also be used in the described studies. The tumor cell line(s) and implantation method that supports the most consistent and reliable tumor growth is used in animal studies testing the lead EGFR nucleic acid(s). Nucleic acids are administered by daily subcutaneous injection or by continuous subcutaneous infusion from Alzet mini osmotic pumps beginning 3 days after tumor implantation and continuing for the duration of the study.
  • Group sizes of at least 10 animals are employed. Efficacy is determined by statistical comparison of tumor volume of nucleic acid-treated animals to a control group of animals freated with saline alone. Because the growth of these tumors is generally slow (45-60 days), an initial endpoint is the time in days it takes to establish an easily measurable primary tumor (i.e. 50-100 mm.3) in the presence or absence of nucleic acid treatment.
  • EGFR e.g., HERl and HER2
  • siNAs against HER2 sites 2344 were tested for the ability to reduce endogenous HER2 RNA and protein in the HER2 overexpressing breast cancer cell line SK-BR-3. Additionally, siNAs were tested for the ability to inhibit proliferation of SK-BR-3 cells. Further, unmodified and additional chemically-modified siNAs (see Table VI) against HER2 site 2344 and 3706 were tested for the ability to reduce endogenous HER2 RNA in the HER2 overexpressing ovarian cancer cell line SK-OV-3 and A549 cells.
  • SK-BR-3 cells were maintained in McCoy's medium (GIBCO/BRL, Bethesda, MD) supplemented with 10% fetal bovine seram, L- glutamine (2 mM), bovine insulin (10 ⁇ g/mL).
  • SK-OV-3 cells were maintained in EMEM medium (GIBCO/BRL, Bethesda, MD) supplemented with 10% fetal bovine seram.
  • RNA and protein endpoints were achieved by the following method: a 5X mixture of siNA (1.95- 250 nM) and a cationic lipid formulation (20 ⁇ g/mL) was made in 150 ⁇ L of growth medium. siNA/lipid complexes were allowed to form for 20 minutes at 37°C under 5% CO 2 .
  • siNA/lipid complexes were left on cells for 24h (RNA endpoint) or 48h (protein endpoint).
  • Real time RT-PCR (Taqman assay) was performed on purified RNA samples using separate primer/probe sets for target HER2 mRNA or control 36B4 RNA. 36B4 RNA levels were used to normalize for differences in well to well sample recovery. RT-PCR conditions were: 30 min at 48°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. Reactions were performed on an ABI Prism 7700 sequence detector.
  • HER2 protein levels were determined by ELISA 48h post-treatment. HER2 protein levels were normalized to cell number (MTS assay) to confrol for differences in well to well sample recovery. Results are shown in Figures 15 and 16 as the average of duplicate treatments ⁇ SD.
  • siNAs for proliferation assays were the same as above except for the following changes. Short pulse transfection and multiple dosing was used, at 24h post- plating 5X siNA/lipid complexes were added to and left on cells for 4h then removed and replaced with growth medium. Final concentration of siNA and inverted controls was 6.25-50 nM. A second dose of siNA lipid was added at 72h post-plating and once again replaced with growth medium after 4h of treatment. Inhibition of cell growth was determined by MTS assay at 48, 72 and 96h post-treatment. Data for the 96h point is shown in Figure 14. Results are shown as the average of triplicate treatments ⁇ SD. As shown in Figure 14, significant inhibition of proliferation is observed using both all RNA and chemically-modified siNA constracts targeting HER2 site 2344 in SKBR-3 cells.
  • RNA expression in A549 cells were plated approximately 24h before transfection in 96-well plates at 5,000-7,500 cells/well, 100 ⁇ l/well, such that at the time of transfection cells are 70-90%> confluent.
  • annealed siNAs were mixed with the fransfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 ⁇ l/well and incubated for 20 min. at room temperature.
  • the siNA transfection mixtures were added to cells to give a final siNA concentration of 25 nM in a volume of 150 ⁇ l.
  • Each siNA fransfection mixture was added to 3 wells for triplicate siNA treatments.
  • RNA polymerase subunit an RNA polymerase subunit
  • a siNA construct comprising ribonucleotides and 3'-terminal dithymidine caps (RPI#30988/31064) was compared to a chemically modified siNA construct comprising 2 '-deoxy-2 '-fluoro pyrimidine nucleotides and purine ribonucleotides in which the sense strand of the siNA is further modified with 5' and 3 '-terminal inverted deoxyabasic caps and the antisense strand comprises a 3'-tenninal phosphorothioate internucleotide linkage (RPI#31300/31301), which was also compared to a matched chemistry inverted confrol (RPI#31312/31313).
  • siNA constracts were also compared to untreated cells, cells transfected with lipid and scrambled siNA constracts (Scraml and Scram2), and cells transfected with lipid alone (transfection control). As shown in the figure, both siNA constructs show significant reduction of EGFR RNA expression. Additional stabilization chemistries as described in Table VIII are similarly assayed for activity.
  • EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • EGFR gene e.g., HERl, HER2, HER3, and/or HER4
  • Particular degenerative and disease states that can be associated with EGFR expression modulation include, but are not limited to, cancer, including breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and any other diseases or conditions that are related to or will respond to the levels of EGFR in a cell or tissue, alone or in combination with other therapies.
  • Herceptin, 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. ribozymes and antisense molecules) of the instant invention.
  • anti-cancer compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) and are hence within the scope of the instant invention.
  • Such compounds and therapies are well l ⁇ iown in the art (see for example Cancer: Principles and Pranctice of Oncology, Volumes 1 and 2, eds Devita, V.T., Hellman, S., and Rosenberg, S.A., J.B.
  • the nucleic acids of the invention are prepared in one of two ways.
  • the agents are physically combined in a preparation of nucleic acid and chemotherapeutic agent, such as a mixture of a nucleic acid of the invention encapsulated in liposomes and ifosfamide in a solution for intravenous administration, wherein both agents are present in a therapeutically effective concentration (e.g., ifosfamide in solution to deliver 1000-1250 mg/n ⁇ 2/day and liposome-associated nucleic acid of the invention in the same solution to deliver 0.1-100 mg/kg/day).
  • the agents are administered separately but simultaneously in their respective effective doses (e.g., about 1000 to about 1250 mg/m2/d ifosfamide and about 0.1 to about 100 mg/kg/day nucleic acid of the invention).
  • Example 11 Diagnostic uses
  • 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.
  • diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example using cellular lysates or partially purified cellular lysates.
  • siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell.
  • the close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA.
  • siNA molecules described in this invention 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).
  • 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.
  • siNA molecules of this invention include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example fluorescence resonance emission transfer (FRET).
  • FRET fluorescence resonance emission transfer
  • 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
  • the second siNA molecules i.e., those that cleave only mutant forms of target RNA
  • 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.
  • 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 is adequate to establish risk.
  • RNA levels are compared qualitatively or quantitatively.
  • LOCUS ERBB2 4530 bp mRNA linear PRI 05-NOV-2002 DEFINITION Homo sapiens v ⁇ erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2) , mRNA.
  • HER3 Homo sapiens v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) (ERBB3) , mRNA. ACCESSION NM_001982 (HER3)
  • HSERB2R (X03363) Human c-erb-B-2 mRNA
  • the 3'-ends of the Upper sequence and the Lower sequence of the siRNA construct can include an overhang sequence, for example 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.
  • NM_005228 Homo sapiens epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) (EGFR), mRNA
  • the 3'-ends of the Upper sequence and the Lower sequence of the siRNA construct can include an overhang sequence, for example 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.
  • 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.
  • Wait time does not include contact time during delivery.
  • Tandem synthesis utilizes double coupling of linker molecule

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US277494 1981-06-26
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US10/163,552 US20030105051A1 (en) 2001-05-29 2002-06-06 Nucleic acid treatment of diseases or conditions related to levels of HER2
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US10/251,117 US20030170891A1 (en) 2001-06-06 2002-09-19 RNA interference mediated inhibition of epidermal growth factor receptor gene expression using short interfering nucleic acid (siNA)
US10/277,494 US20030186909A1 (en) 1997-01-27 2002-10-21 Nucleic acid treatment of diseases or conditions related to levels of epidermal growth factor receptors
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