WO2012018881A2 - Methods and compositions for the regulation of rna - Google Patents

Methods and compositions for the regulation of rna Download PDF

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Publication number
WO2012018881A2
WO2012018881A2 PCT/US2011/046365 US2011046365W WO2012018881A2 WO 2012018881 A2 WO2012018881 A2 WO 2012018881A2 US 2011046365 W US2011046365 W US 2011046365W WO 2012018881 A2 WO2012018881 A2 WO 2012018881A2
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protein
incrna
cell
nucleotides
dsrna
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PCT/US2011/046365
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French (fr)
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WO2012018881A3 (en
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Kenneth S. Koblan
Stuart Pollard
Gregory Hinkle
Brian Bettencourt
Donna T. Ward
Muthiah Manoharan
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Alnylam Pharmaceuticals, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids

Definitions

  • the invention relates to the regulation of non-coding RNAs, specifically long intervening non-coding RNA (IncRNA) genes and transcripts.
  • IncRNA non-coding RNA
  • Non-coding RNA (ncRNA) genes produce functional RNA molecules rather than encoding proteins.
  • ncRNA genes have been poorly characterized and only recently the topic of serious inquiry.
  • Several ncRNA genes have recently been identified and these appear to have diverse roles in cellular regulation and gene expression as structural, catalytic and regulatory molecules (See Eddy S. Curr Opin Genet Devel, 9: 695-699, 1999; Eddy, S. Nature Reviews, Genetics, 2: 919-929, 2001; and Mattick, J., et al., Mol. Biol. & Evolution, vol. 18, no. 9, 16: 1611-1630, 2001).
  • CNS noncoding sequences
  • ncRNA The most prominent and well-studied example of a functional ncRNA in the literature to date is the ncRNA, XIST (Plath, et al, Science, 300; 131-135; 2003).
  • Rinn and colleages identified the gene known as HOTAIR, which represses transcription in trans across the HOXD cluster, suggesting that ncRNA may play a critical role in chromosomal demarcation and chromatin states that affect cancer metastasis (Rinn, et al, Cell, 129(7); 131 1-1323; 2007; Gupta, R., et al, Nature Letters, 464, 1071; 2010; See also Koziol and Rinn, Curr. Opinion in Gen. Dev., 20, 142-148; 2010).
  • lincRNAs contain features of normal coding genes including promoters and a multi-exonic structure (Guttman, M., et al., Nature Biotech., 1-8, 2010). While this work was facilitated by the creation of whole transcriptome libraries of mouse and human tissues (Okazaki, Y., et al., Nature, 420: 563-573, 2002), much is still unknown about the large category of non-coding RNA genes, their transcripts and role in gene regulation and cell biology.
  • NATs natural antisense transcripts
  • pseudogenes have been shown to have coding-independent functions (Poliseno, L., et al, Nature, 465: 1033-1038, 2010); and NATs have been investigated for their role in the development and role in the nervous system and as potential drug targets (Wahlestedt, C, Drug Discovery Today, 1 1 : 503-508; 2006; Faghihi, M., and Wahlstedt, C, Genome Biology, 7:R38, 2006; St. Laurent, G., and Wahlstedt, C, TRENDS in Neuro., 30: 612-621; Faghihi, M., et al, Nat. Med.,
  • compositions and methods of modulation the status, activity, or expression of long intervening (which includes both intronic and intergenic) non-coding RNAs (IncRNAs) in a cell, tissue or organism are also provided.
  • compositions and methods for treating pathological conditions and diseases in a mammal caused by or modulated by the regulatory, structural, catalytic or signaling properties of a lncRNA are further disclosed. diagnostic methods, kits and assays which are designed to utilize the cellular pathways and systems associated with lncRNA targets.
  • Targets of the present invention include long intervening (intronic and intergenic) non-coding RNAs, or "IncRNAs", also known in the art as macroRNAs and efference RNAs (eRNAs).
  • IncRNAs also known in the art as macroRNAs and efference RNAs (eRNAs).
  • eRNAs efference RNAs
  • the term “lncRNA”, or “long intervening non-coding RNA” refers broadly to the targets of the present invention and include the “IncRNA gene”, as well as the resultant "IncRNA transcript.”
  • the family of IncRNA targets envisioned by the present inventors also includes NATs (Natural Antisense Transcripts).
  • pseudogenes are also considered to fall within the broader family of IncRNA targets of the present invention.
  • IncRNA genes of the present invention are processed to produce “IncRNA transcripts” and these transcripts may be transcribed from either strand of the chromosomal DNA.
  • the term “IncRNA” refers broadly to the IncRNA gene, as well as the resultant IncRNA transcript.
  • IncRNA genes may be as small as lkb (kilobase) or as large as lOOkb (kilobases) while IncRNA transcripts may range in size from 200 nucleotides to 20kb.
  • IncRNA transcripts may be at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, at least 5000 nucleotides, at least 10,000 nucleotides or at least 20,000 nucleotides, and range from 250-300 nucleotides, 300- 400 nucleotides, 400-500 nucleotides, 500-600 nucleotides, 600-700 nucleotides, 700-800 nucleotides, 800-900 nucleotides, 900-1000 nucleotides, 1000-5000 nucleotides, 5000-10,000 nucleotides, or 10,000-20,000 nucleotides in length.
  • the term "IncRNA gene” refers to the IncRNA which is encoded within the genome or in a genomic construct (whether natural or synthetic) and has at least one feature of a coding gene selected from the group including, but not limited to, (i) a promoter or promoter-like feature such as one or more proximal regulatory elements; (ii) one or more exons; (iii) a polyA signature and (iv) which encodes a transcribed RNA, i.e., a IncRNA transcript.
  • Endogenous IncRNA genes e.g., those encoded within or engineered to be encoded by a host cell genome, are characterized by their intervening genomic location. This means that endogenous or wildtype IncRNAs may be intronic or intergenic.
  • IncRNAs are those found to be encoded substantially within an intron of a gene.
  • Intergenic IncRNAs are those found to be encoded between two different genes.
  • the term "IncRNA transcript” refers to an RNA transcript encoded by a IncRNA gene and which is (i) at least 200 nucleotides in length and (ii) does not encode a mature or complete protein product.
  • IncRNA transcripts may encode peptides of 50 amino acids or less. It should be understood that IncRNA transcripts may be synthesized as IncRNA transcript variants, which may be engineered to encode peptides or proteins.
  • Other types of large non-coding RNA transcripts found in cells such as primary microRNAs (pri-miRs) which are processed to produce micro-RNAs or small RNA species are not targets of the present invention.
  • Intervening when used in the context of IncRNAs means intronic or intergenic.
  • IncRNA genes and transcript targets are listed in Table 2.
  • IncRNA genes were identified from the Ensembl database (www.ensembl.org) and their respective RNA transcripts were extracted.
  • Table 2 lists the Ensembl gene identifier of each IncRNA gene, (prefix ENSG), each IncRNA transcript (prefix ENST), in addition to the human chromosomal location of each gene (column 1, designated "C").
  • Homo_sapiens.GRCh37.58.gif i.e., annotation version 58 from the Ensembl database.
  • the sequence as well as other characteristics of each upstream and downstream gene identified may be identified by searching the Ensembl database or the NCBI database using the unique identifiers reported in Table 2.
  • IncRNA intronic, i.e., found encoded within an intron of a coding gene.
  • LDAs lncRNA-directed agents
  • IncRNA expression pattern or activity can be altered.
  • Protein coding genes found upstream of IncRNA genes or transcripts may be targeted by any of the methods disclosed herein. These genes include, but are not limited to, 2',3'-cyclic nucleotide 3' phosphodiesterase; 3-phosphoinositide dependent protein kinase-1 ; 5'-nucleotidase domain containing 2; 5 '-nucleotidase, cytosolic II; A kinase (PRKA) anchor protein 1 1; A kinase (PRKA) anchor protein 8-like; abhydrolase domain containing 6; acetyl-CoA acyltransferase 2; acetyl-CoA carboxylase alpha; activating transcription factor 7 interacting protein 2; acyl-CoA oxidase 1, palmitoyl; acyl-CoA synthetase bubblegum family member 2; acyl-CoA synthetase long-chain family member 3; acyl-Co
  • alkylglycerone phosphate synthase alpha 1,4- galactosyltransferase
  • angiopoietin 2 ankyrin 3, node of Ranvier (ankyrin G)
  • ankyrin repeat and SOCS box-containing 3 ankyrin repeat and SOCS box-containing 7
  • ankyrin repeat and sterile alpha motif domain containing IB ankyrin repeat domain 11 ; annexin A4; antigen p97
  • ATP binding domain 4 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1; ATPase, Ca++ transporting, type 2C, member 2; ATPase, class VI, type 1 1A; ATPase, H+ transporting VO subunit e2; ATPase, H+ transporting, lysosomal 3 IkDa, VI subunit E2; ATPase, H+ transporting, lysosomal 42kDa, VI subunit CI ; ATPase, H+
  • coli bone marrow stromal cell antigen 2
  • bone morphogenetic protein 8a brain and acute leukemia, cytoplasmic; brain and reproductive organ-expressed (TNFRSF1A modulator); breast carcinoma amplified sequence 3; brevican; BRF2, subunit of RNA polymerase III transcription initiation factor, BRF 1 -like; BRI3 binding protein;
  • bromodomain PHD finger transcription factor; BTB and CNC homology 1 basic leucine zipper transcription factor 2; butyrophilin, subfamily 2, member Al; C2 calcium-dependent domain containing 4B; cadherin 10, type 2 (T2-cadherin); cadherin 11, type 2, OB-cadherin (osteoblast); cadherin 18, type 2; cadherin 20, type 2; cadherin 5, type 2 (vascular endothelium); cadherin 9, type 2 (Tl -cadherin); calbindin 1, 28kDa; calcineurin binding protein 1; calcium channel, voltage-dependent, L type, alpha 1C subunit; calcium homeostasis endoplasmic reticulum protein; caldesmon 1 ; cAMP responsive element binding protein 5; cancer susceptibility candidate 5; cancer/testis antigen family 47, member Al; CAP, adenylate cyclase-associated protein, 2 (yeast); caprin family member 2; carbohydrate (chondroitin
  • carboxylesterase 7 ; carboxylesterase 7; carboxypeptidase A4; carboxypeptidase A5;
  • carboxypeptidase E carnitine palmitoyltransferase 1A (liver); carnosine dipeptidase 1
  • cell division cycle 73 Pafl/RNA polymerase II complex component, homolog (S. cerevisiae); cell division cycle and apoptosis regulator 1 ; cell division cycle associated 4; cellular repressor of ElA-stimulated genes 1; CGRP receptor component;
  • chemokine (C-C motif) ligand 15 chemokine (C-C motif) ligand 4-like 1; chemokine (C-C motif) ligand 5; chemokine (C-X-C motif) ligand 2; chemokine (C-X-C motif) receptor 2; chemokine (C-X-C motif) receptor 3; chemokine (C-X- C motif) receptor 5; chemokine-like receptor 1 ; cholinergic receptor, nicotinic, alpha 4; chondrolectin; chromobox homolog 4 (Pc class homolog, Drosophila); chromobox homolog 7; chromodomain helicase DNA binding protein 4; chromosome 1 open reading frame 124;
  • chromosome 1 open reading frame 151 chromosome 1 open reading frame 43; chromosome 1 open reading frame 86; chromosome 10 open reading frame 114; chromosome 10 open reading frame 71; chromosome 1 1 open reading frame 2; chromosome 1 1 open reading frame 34;
  • congenital dyserythropoietic anemia type I; consortin, connexin sorting protein; contactin 5; contactin associated protein-like 2; contactin associated protein-like 3B; contactin associated protein-like 4; copper metabolism (Murrl) domain containing 1 ; core-binding factor, runt domain, alpha subunit 2; translocated to, 3; corticotropin releasing hormone; CREB regulated transcription coactivator 3; Crm, cramped-like (Drosophila); CTD (carboxy -terminal domain, R A polymerase II, polypeptide A) small phosphatase 2; CTP synthase; C-type lectin domain family 4, member M; CUE domain containing 2; CUGBP, Elav-like family member 4; cullin- associated and neddylation-dissociated 1; cyclin-dependent kinase 1 1 A; cyclin-dependent kinase 6; cylicin, basic protein of sperm
  • cerevisiae -like; discs, large (Drosophila) homolog-associated protein 1 ; disrupted in renal carcinoma 2; DnaJ (Hsp40) homolog, subfamily C, member 5 gamma; dopey family member 1; DOT 1 -like, histone H3 methyltransferase (S. cerevisiae); doublecortin-like kinase 1; doublesex and mab-3 related transcription factor 3; Down syndrome cell adhesion molecule like 1; dpy-19-like 1 (C.
  • elegans drebrin 1; dual specificity phosphatase 10; dual specificity phosphatase 4; dynactin 5 (p25); dynactin 6; dynein, cytoplasmic 1, light intermediate chain 2; dynein, light chain, LC8-type 1 ; dynein, light chain, roadblock-type 2; dystroglycan 1
  • E2F transcription factor 2 E74-like factor 1 (ets domain transcription factor); early B-cell factor 1 ; early B-cell factor 3; echinoderm microtubule associated protein like 2; echinoderm microtubule associated protein like 5; echinoderm microtubule associated protein like 6; ECSIT homolog (Drosophila); egf-like module containing, mucin-like, hormone receptor-like 2; EH-domain containing 4; elastin microfibril interfacer 3; ELKS/RAB6-interacting/CAST family member 1 ; elongation factor 1 homolog (S.
  • elongation factor Tu GTP binding domain containing 1 embryonic ectoderm development; endonuclease domain containing 1 ; endoplasmic reticulum-golgi intermediate compartment (ERGIC) 1 ; endosulfine alpha; endothelial cell adhesion molecule; enhancer of rudimentary homolog (Drosophila); enolase superfamily member 1; erythrocyte membrane protein band 4.1 like 5; eukaryotic translation initiation factor 4 gamma, 2; eukaryotic translation initiation factor 4B; even-skipped homeobox 1 ; exocyst complex component 4; exosome component 3; exportin 6; family with sequence similarity 105, member A; family with sequence similarity 111, member B; family with sequence similarity 113, member B; family with sequence similarity 125, member B; family with sequence similarity 126, member A; family with sequence similarity 156, member B; family with sequence similarity
  • fucosyltransferase 3 galactoside 3(4)-L-fucosyltransferase, Lewis blood group
  • fumarylacetoacetate hydrolase (fumarylacetoacetase); furin (paired basic amino acid cleaving enzyme); FY oncogene related to SRC, FGR, YES; G kinase anchoring protein 1; G protein regulated inducer of neurite outgrowth 1; G protein-coupled estrogen receptor 1; G protein- coupled receptor 107; G protein-coupled receptor 176; G protein-coupled receptor 180; G protein-coupled receptor 20; G protein-coupled receptor 81; GA binding protein transcription factor, beta subunit 1 ; galanin receptor 1; gamma-glutamyltransferase light chain 2; gap junction protein, delta 2, 36kDa; GATA zinc finger domain containing 2B; GDP-mannose 4,6- dehydratase; gelsolin; general transcription factor IIA, 1, 19/37kDa; general transcription factor IIH, polypeptide 2, 44kDa; general transcription factor IIH, polypeptide 2C; general transcription factor IIH, polypeptide 5; germ cell associated
  • hexosaminidase A (alpha polypeptide); high mobility group nucleosomal binding domain 3; high mobility group nucleosomal binding domain 4; histamine receptor H4; histone cluster 1, H3f; histone cluster 1, H4i; histone cluster 4, H4; homeobox A6; homeobox B8; homeobox containing 1; homeodomain interacting protein kinase 2; homer homolog 2 (Drosophila); homer homolog 3 (Drosophila); hyaluronan synthase 2; hyaluronan-mediated motility receptor (RHAMM);
  • hydroxyacid oxidase 2 long chain
  • hydroxysteroid (17-beta) dehydrogenase 12 hydroxysteroid (17-beta) dehydrogenase 12
  • IMP inosine 5 '-monophosphate dehydrogenase 1
  • importin 5 ⁇ 80 homolog
  • inositol 1,4,5- triphosphate receptor interacting protein inositol 1,4,5 -triphosphate receptor, type 3; inositol hexakisphosphate kinase 2; insulin induced gene 2; integrin, alpha D; integrin, alpha E (antigen CD 103, human mucosal lymphocyte antigen 1 ; alpha polypeptide); integrin, beta 3 (platelet glycoprotein Ilia, antigen CD61); interferon induced transmembrane protein 1 (9-27); interferon regulatory factor 2; interferon regulatory factor 8; interleukin 12 receptor, beta 2; interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40);
  • interleukin 17C interleukin 21 receptor; interleukin 22; intermediate filament tail domain containing 1 ; intracisternal A particle-promoted polypeptide; IQ motif and Sec7 domain 1 ; IQ motif and Sec7 domain 2; IQ motif and Sec7 domain 3; IQ motif containing H; jagged 1 (Alagille syndrome); kaptin (actin binding protein); kelch repeat and BTB (POZ) domain containing 13; kelch repeat and BTB (POZ) domain containing 6; kelch-like 12 (Drosophila); kelch-like 28 (Drosophila); kelch-like 36 (Drosophila); keratin 73; KIAA0020; KIAA0101; KIAA0125; KIAA0174; KIAA0240; KIAA0355; KIAA0415; KIAA0564; KIAA0776;
  • lipase maturation factor 1 lipase, hepatic; lipocalin 6; Ion peptidase 2, peroxisomal; loss of heterozygosity, 12, chromosomal region 1; LSM11, U7 small nuclear RNA associated; LSM7 homolog, U6 small nuclear RNA associated (S. cerevisiae); LY6/PLAUR domain containing 5; lymphocyte antigen 86;
  • lymphocyte cytosolic protein 2 (SH2 domain containing leukocyte protein of 76kDa); lysine (K)- specific demethylase 3 A; lysozyme-like 1; macrophage scavenger receptor 1; mahogunin, ring finger 1; major histocompatibility complex, class I, G; major histocompatibility complex, class II, DM alpha; mannosidase, alpha, class 1A, member 1 ; mannosidase, beta A, lysosomal;
  • mannosyl alpha-l,3-)-glycoprotein beta-l,2-N-acetylglucosaminyltransferase; mannosyl (beta- l,4-)-glycoprotein beta-l,4-N-acetylglucosaminyltransferase; mastermind-like 3 (Drosophila); matrilin 2; matrix metallopeptidase 16 (membrane-inserted); MAX dimerization protein 1 ; MAX gene associated; mediator complex subunit 13-like; mediator complex subunit 25; mediator complex subunit 27; mediator complex subunit 6; Mediterranean fever; melanoma cell adhesion molecule; membrane associated guanylate kinase, WW and PDZ domain containing 2; menage a Peru homolog 1, cyclin H assembly factor (Xenopus laevis); mesencephalic astrocyte-derived neurotrophic factor; mesoderm specific transcript homolog (mouse); metadherin; met
  • microsomal glutathione S-transferase 3 microtubule associated serine/threonine kinase 2; microtubule associated tumor suppressor candidate 2; microtubule- associated protein 1 light chain 3 beta 2; midnolin; mindbomb homolog 1 (Drosophila);
  • minichromosome maintenance complex component 8 mitochondrial ribosomal protein 63; mitochondrial ribosomal protein L41 ; mitochondrial ribosomal protein S I 1 ; mitochondrial ribosomal protein S25; mitochondrial translational release factor 1 ; mitogen-activated protein kinase 13; mitogen-activated protein kinase kinase 6; mitogen-activated protein kinase kinase kinase 14; MON2 homolog (S.
  • neurocanthocytosis neuron navigator 3; neuropeptide FF-amide peptide precursor; neuropilin (NRP) and tolloid (TLL)-like 1 ; neurotrophic tyrosine kinase, receptor, type 3; NFKB activating protein; NHS-like 1 ; nicotinamide nucleotide adenylyltransferase 3; NIMA (never in mitosis gene a)-related kinase 5; nitric oxide synthase 2, inducible; NK2 transcription factor related, locus 5 (Drosophila); NLR family, pyrin domain containing 5; NMDA receptor regulated 2; N- myc (and STAT) interactor; N-myristoyltransferase 2; non-SMC element 1 homolog (S.
  • nuclear receptor binding protein 2 nuclear receptor coactivator 2; nuclear receptor subfamily 1, group D, member 2; nuclear receptor subfamily 4, group A, member 2; nucleolin; nucleoporin 210kDa-like; nucleoporin 214kDa; NudC domain containing 1; nudix (nucleoside diphosphate linked moiety X)-type motif 11 ; numb homolog (Drosophila); odz, odd Oz/ten-m homolog 4 (Drosophila); olfactory receptor, family 4, subfamily F, member 29; olfactory receptor, family 4, subfamily F, member 4; olfactory receptor, family 4, subfamily F, member 5; olfactory receptor, family 5, subfamily AU, member 1 ; outer dense fiber of sperm tails 3-like 1 ; paired box 6; pantothenate kinase 2; papilin, proteoglycan-like sulfated
  • peptidylprolyl isomerase A (cyclophilin A)-like 4B; perforin 1 (pore forming protein); pericentrin; periphilin 1 ; peroxisome proliferator-activated receptor alpha; peroxisome proliferator-activated receptor gamma, coactivator 1 beta; PHD finger protein 12; PHD finger protein 3; phenylalanyl-tRNA synthetase 2, mitochondrial; phosphatase and actin regulator 4; phosphatase and tensin homolog; phosphatidylethanolamine-binding protein 4;
  • phosphatidylinositol binding clathrin assembly protein phosphatidylinositol binding clathrin assembly protein; phosphatidylinositol glycan anchor biosynthesis, class L; phosphatidylinositol glycan anchor biosynthesis, class W;
  • phosphatidylinositol transfer protein membrane-associated 2; phosphatidylinositol-3,4,5- trisphosphate-dependent Rac exchange factor 1; phosphatidylinositol-3,4,5-trisphosphate- dependent Rac exchange factor 2; phosphatidylinositol-4-phosphate 5-kinase, type I, beta;
  • phosphatidylinositol-4-phosphate 5-kinase type I, gamma; phosphoglucomutase 5;
  • phosphoinositide-3-kinase class 2, alpha polypeptide; phospholipase A2, group IVC (cytosolic, calcium-independent); phospholipase A2, group XIIB; phosphomannomutase 1; phosphorylase kinase, alpha 2 (liver); phosphorylase, glycogen, liver; phytanoyl-CoA dioxygenase domain containing 1 ; pituitary tumor-transforming 1 interacting protein; plasminogen-like B 1 ; platelet- activating factor acetylhydrolase lb, regulatory subunit 1 (45kDa); pleckstrin homology domain containing, family A member 5; pleckstrin homology domain containing, family G (with RhoGef domain) member 6; pleckstrin homology domain containing, family O member 1 ; pleomorphic adenoma gene-like 1 ; PMS1 postmeiotic segregation increased 1 (S.
  • polyamine modulated factor 1 binding protein 1 polybromo 1; polymerase (RNA) II (DNA directed) polypeptide J2; polymerase (RNA) II (DNA directed) polypeptide J3; polymerase (RNA) III (DNA directed) polypeptide B; polymerase (RNA) III (DNA directed) polypeptide F, 39 kDa; post-GPI attachment to proteins 2; potassium channel modulatory factor 1 ; potassium channel tetramerisation domain containing 13; potassium large conductance calcium-activated channel, subfamily M, alpha member 1 ; potassium large conductance calcium-activated channel, subfamily M, beta member 4; POU class 2 associating factor 1; POU class 2 homeobox 2; PR domain containing 11 ; PR domain containing 4; pre-B-cell leukemia homeobox 4; pregnancy- zone protein; presenilin enhancer 2 homolog (C.
  • proenkephalin progesterone receptor membrane component 2; progestin and adipoQ receptor family member V; progestin and adipoQ receptor family member VI; prolactin; proline rich 7 (synaptic); proline rich Gla (G- carboxyglutamic acid) 4 (transmembrane); proline-rich protein BstNI subfamily 2; prolyl 4- hydroxylase, alpha polypeptide I; prolyl 4-hydroxylase, alpha polypeptide III; prostaglandin F receptor (FP); prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase); protease, serine, 1 (trypsin 1); protease, serine, 12 (neurotrypsin, motopsin); proteasomal ATPase-associated factor 1; proteasome (prosome, macropain) subunit, alpha type, 5; proteasome (prosome,
  • protein phosphatase 1, regulatory (inhibitor) subunit 3B protein phosphatase 2, regulatory subunit B, alpha; protein phosphatase 2, regulatory subunit B", gamma; protein phosphatase 3, catalytic subunit, gamma isozyme; protein phosphatase 5, catalytic subunit; protein phosphatase, Mg2+/Mn2+ dependent, 1H; protein tyrosine phosphatase, non-receptor type 23; protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 1 ; protein tyrosine phosphatase, receptor type, H; protocadherin alpha 2; protocadherin beta 1;
  • coli (S. cerevisiae); RAD52 homolog (S. cerevisiae); Ral GTPase activating protein, alpha subunit 1 (catalytic); Rap guanine nucleotide exchange factor (GEF) 3; RAPIB, member of RAS oncogene family; Ras association (RalGDS/AF-6) domain family ( -terminal) member 7; ras homolog gene family, member Q; RAS protein activator like 2; receptor accessory protein 3; regenerating islet-derived family, member 4; regulator of chromosome condensation 1; regulator of chromosome condensation 2; regulator of G-protein signaling 20; regulator of G-protein signaling 7; regulator of G-protein signaling 8; regulatory associated protein of MTOR, complex 1 ; reprimo-like; resistance to inhibitors of cholinesterase 3 homolog (C.
  • GEF Rap guanine nucleotide exchange factor
  • elegans retinoblastoma binding protein 6; retinoic acid receptor, alpha; REV3- like, catalytic subunit of DNA polymerase zeta (yeast); REX1, RNA exonuclease 1 homolog (S. cerevisiae); REX1, RNA exonuclease 1 homolog (S.
  • Rho GTPase activating protein 17 Rho GTPase activating protein 17; Rho GTPase activating protein 22; Rho GTPase activating protein 28; rhomboid 5 homolog 2 (Drosophila); Rho-related BTB domain containing 2; ribonuclease HI ; ribonuclease P/MRP 40kDa subunit; ribonucleoprotein, PTB-binding 2; ribonucleotide reductase M2 B (TP53 inducible); ribosomal modification protein rimK-like family member B; ribosomal protein L14; ribosomal protein L31 ; ribosomal protein L36a-like; ribosomal protein L7; ribosomal protein SA pseudogene 58; ribosomal protein, large, PI ; ring finger protein 135; ring finger
  • RPGRIP 1 -like RUN and FYVE domain containing 3; RUN domain containing 2C; runt-related transcription factor 2; SI 00 calcium binding protein Al l; SI 00 calcium binding protein A4; SAFB-like, transcription modulator; sal-like 1
  • SH3 and PX domains 2 A SH3 domain containing 19; SHC (Src homology 2 domain containing) family, member 4; short chain dehydrogenase/reductase family 39U, member 1; sialic acid binding Ig-like lectin 5; sideroflexin 5; signal peptidase complex subunit 2 homolog (S. cerevisiae); signal recognition particle 14kDa (homologous Alu RNA binding protein); SIK family kinase 3; sine oculis binding protein homolog (Drosophila); SLAM family member 7; SMAD family member 6; small G protein signaling modulator 3; small nuclear
  • solute carrier family 10 sodium/bile acid cotransporter family
  • member 5 solute carrier family 12
  • solute carrier family 16 sodium/potassium/chloride transporters
  • solute carrier family 16 member 1 (monocarboxylic acid transporter 1); solute carrier family 16, member 3 (monocarboxylic acid transporter 4); solute carrier family 16, member 4 (monocarboxylic acid transporter 5); solute carrier family 17, member 9; solute carrier family 2 (facilitated glucose transporter), member 11 ; solute carrier family 2 (facilitated glucose transporter), member 9; solute carrier family 22, member 23; solute carrier family 22, member 23; solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3; solute carrier family 25, member 13 (citrin); solute carrier family 25, member 30; solute carrier family 26 (sulfate transporter), member 2; solute carrier family 27 (fatty acid transporter), member 5; solute carrier family 35, member B3; solute carrier family 35, member F5; solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; solute carrier family 39 (zinc transporter), member 14
  • synaptotagmin VII synaptotagmin X
  • syntaxin 4 syntaxin binding protein 5 (tomosyn);
  • TBP TATA box binding protein
  • tectonin beta-propeller repeat containing 2; testis expressed 10; tetraspanin 1 1; tetraspanin 4; tetraspanin 9; tetratricopeptide repeat domain 23-like; tetratricopeptide repeat domain 39B; tetratricopeptide repeat domain 4; tetratricopeptide repeat domain 7B; tetratricopeptide repeat domain 8; TGFB-induced factor homeobox 2; THO complex 2; thrombospondin 2; thyrotropin- releasing hormone degrading enzyme; tight junction protein 2 (zona occludens 2); TNF receptor- associated factor 5; toll-like receptor 1 ; topoisomerase (DNA) I, mitochondrial; torsin A interacting protein 2; tousled-like kinase 1 ; tousled-like kinase 2; TOX high mobility group box family member 3; TRAF3 interacting protein 2; trafficking protein particle complex 9;
  • transcription elongation factor A (SII), 1 ; transcription elongation factor A (Sll)-like 1 ;
  • transcription factor 12 transcription factor 4; transcription factor 7-like 1 (T-cell specific, HMG- box); transcription factor Dp-1 ; transducin (beta)-like 1 X-linked receptor 1; transducin-like enhancer of split 3 (E(spl) homolog, Drosophila); translocase of inner mitochondrial membrane 23 homolog B (yeast); translocation associated membrane protein 1 ; transmembrane 6 superfamily member 2; transmembrane and coiled-coil domain family 1 ; transmembrane and coiled-coil domains 4; transmembrane and coiled-coil domains 5A; transmembrane anterior posterior transformation 1; transmembrane protease, serine 9; transmembrane protein 121 ;
  • transmembrane protein 121 transmembrane protein 121 ; transmembrane protein 132B; transmembrane protein 132D;
  • transmembrane protein 14C transmembrane protein 14C; transmembrane protein 154; transmembrane protein 164;
  • transmembrane protein 196 transmembrane protein 2; transmembrane protein 30B;
  • transmembrane protein 59 transmembrane protein 64; transmembrane protein 90B; transportin 3; tribbles homolog 1 (Drosophila); trichorhinophalangeal syndrome I; trinucleotide repeat containing 18; TRIO and F-actin binding protein; tripartite motif-containing 16; tripartite motif- containing 36; tripartite motif-containing 4; tRNA methyltransferase 12 homolog (S. cerevisiae); tRNA splicing endonuclease 2 homolog (S.
  • tropomodulin 3 ubiquitous
  • troponin I type 3 cardiac
  • TSPY-like 2 tubby homolog (mouse); tuberous sclerosis 1; tubulin, alpha lc; tubulin, alpha lc; tubulin, beta 2A; tubulin, beta 2B; tumor necrosis factor (ligand) superfamily, member 4; tumor necrosis factor receptor superfamily, member 10a; tumor necrosis factor receptor superfamily, member 10b; tumor necrosis factor, alpha-induced protein 2; tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; tyrosine kinase, non-receptor, 2; tyrosylprotein sulfotransferase 1; UBA domain containing 1 ; UBA domain containing 2; ubiquitin fusion degradation 1 like (yeast); ubiquitin protein ligase E3 component
  • vacuolar protein sorting 18 homolog (S. cerevisiae); vascular endothelial growth factor A;
  • vascular endothelial zinc finger 1 vasohibin 1 ; vestigial like 3 (Drosophila); vestigial like 4 (Drosophila); v-ets erythroblastosis virus E26 oncogene homolog 1 (avian); vinculin; vitamin K epoxide reductase complex, subunit 1 -like 1 ; vitelline membrane outer layer 1 homolog
  • v-maf musculoaponeurotic fibrosarcoma oncogene homolog G avian
  • v-myc myelocytomatosis viral oncogene homolog avian
  • von Hippel-Lindau tumor suppressor von Willebrand factor
  • v-ral simian leukemia viral oncogene homolog A ras related
  • v-rel reticuloendotheliosis viral oncogene homolog (avian); WAS protein family homolog 2 pseudogene; WAS protein homolog associated with actin, golgi membranes and microtubules; WD repeat domain 44; WD repeat domain 70; Werner helicase interacting protein 1 ; wingless- type MMTV integration site family, member 1 ; wingless-type MMTV integration site family, member 9A; WW domain binding protein 4 (formin binding protein 21); WWC family member 3; XK, Kell blood group complex subunit-related family, member 5;
  • IncRNA transcripts or genes found at or near a chromosomal locus associated with malignancy are targeted for the treatment, diagnosis or therapeutic outcomes.
  • IncRNA transcripts found downstream of the coding gene, septin 9 may be targeted in the treatment or diagnosis of cancers such as ovarian cancer.
  • IncRNA transcripts are encoded between two coding genes known to have an association with malignancy or disease and these are preferred targets of the invention. For example, several IncRNA genes are found coded between v-myc genes and gasdermin C, a coding gene whose product is associated with metastatic melanoma. [0032] In one embodiment, IncRNA transcripts or genes are encoded either upstream or downstream (nearest neighbor) to one or more pseudogenes. These IncRNA are also preferred targets for the therapeutic outcomes described herein.
  • Protein coding genes found downstream of IncRNA genes or transcripts may be targeted by any of the methods disclosed herein. These genes include, but are not limited to 1- acylglycerol-3 -phosphate O-acyltransferase 5 (lysophosphatidic acid acyltransferase, epsilon); 1- aminocyclopropane-l-carboxylate synthase homolog (Arabidopsis)(non-functional)-like; 3- hydroxyisobutyrate dehydrogenase; 3-phosphoinositide dependent protein kinase- 1 ; A kinase (PRKA) anchor protein 11 ; A kinase (PRKA) anchor protein 8-like; abhydrolase domain containing 6; acetoacetyl-CoA synthetase; acid phosphatase 5, tartrate resistant; acidic (leucine- rich) nuclear phosphoprotein 32 family, member E; aconitase 2, mitochondrial; actin related
  • adaptor-related protein complex 3 beta 2 subunit; adaptor-related protein complex 4, epsilon 1 subunit; additional sex combs like 1 (Drosophila); ADP-ribosylation factor-like 17A; ADP- ribosylation factor-like 5B; adrenergic, alpha- 1B-, receptor; AF4/FMR2 family, member 1; aldehyde dehydrogenase 1 family, member A3 ; aldo-keto reductase family 1 , member C 1 (dihydrodiol dehydrogenase 1 ; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase); alkB, alkylation repair homolog 5 (E.
  • alpha 1,4-galactosyltransferase alpha-2-macroglobulin; alpha-2-macroglobulin-like 1; amino-terminal enhancer of split; amphiphysin; amyloid beta (A4) precursor protein; amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein; angel homolog 1 (Drosophila); ankyrin 3, node of Ranvier (ankyrin G); ankyrin repeat and sterile alpha motif domain containing IB; ankyrin repeat domain 1 1; ankyrin repeat domain 20 family, member A3; ankyrin repeat domain 42; ankyrin repeat domain 50; annexin A4;
  • MDR/TAP member 1
  • ATP-binding cassette sub-family C
  • CTR/MRP sub-family C
  • ATX1 antioxidant protein 1 homolog yeast
  • AU R A binding protein/enoyl-CoA hydratase; B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB; BAH domain and coiled-coil containing 1 ; BAI1 -associated protein 2-like 1 ; basic helix-loop-helix family, member e23; basonuclin 1 ; bassoon (presynaptic cytomatrix protein); BCDIN3 domain containing; B-cell translocation gene 4; Bcl2 modifying factor; BCL6 corepressor; beta-l,4-N- acetyl-galactosaminyl transferase 4; Bloom syndrome, RecQ helicase-like; bobby sox homolog (Drosophila); bol, boule-like (Drosophila); bradykinin receptor B
  • cell division cycle and apoptosis regulator 1 cell division cycle and apoptosis regulator 1 ; cellular retinoic acid binding protein 2; centromere protein V; ceramide kinase; cerebellin 2 precursor; chemokine (C- C motif) ligand 15; chemokine (C-X-C motif) receptor 5; chloride intracellular channel 5;
  • CHMP family member 7; chondrolectin; chromatin modifying protein 2B; chromobox homolog 7; chromodomain helicase DNA binding protein 3; chromodomain helicase DNA binding protein 4; chromodomain helicase DNA binding protein 6; chromodomain protein, Y-like 2;
  • chromosome 1 open reading frame 222 chromosome 1 open reading frame 43; chromosome 1 open reading frame 97; chromosome 10 open reading frame 11 ; chromosome 10 open reading frame 140; chromosome 1 1 open reading frame 2; chromosome 1 1 open reading frame 61 ; chromosome 1 1 open reading frame 73; chromosome 12 open reading frame 49; chromosome 12 open reading frame 70; chromosome 13 open reading frame 28; chromosome 14 open reading frame 104; chromosome 14 open reading frame 126; chromosome 14 open reading frame 145; chromosome 14 open reading frame 153; chromosome 14 open reading frame 159; chromosome 14 open reading frame 177; chromosome 14 open reading frame 182; chromosome 14 open reading frame 183; chromosome 15 open reading frame 41 ; chromosome 15 open reading frame 54; chromosome 15 open reading frame 57; chromosome 16 open reading frame 89;
  • chromosome 17 open reading frame 108 chromosome 17 open reading frame 57; chromosome 17 open reading frame 72; chromosome 17 open reading frame 82; chromosome 19 open reading frame 23; chromosome 19 open reading frame 42; chromosome 2 open reading frame 40;
  • chromosome 9 open reading frame 30 chromosome 9 open reading frame 93; chronic lymphocytic leukemia up-regulated 1 opposite strand; chymase 1, mast cell; cingulin-like 1 ; clathrin interactor 1; claudin 16; coactosin-like 1 (Dictyostelium); coatomer protein complex, subunit epsilon; COBW domain containing 3; coenzyme Q9 homolog (S.
  • coiled-coil domain containing 102B coiled-coil domain containing 147; coiled-coil domain containing 155; coiled-coil domain containing 28A; coiled-coil domain containing 8; collagen, type XXIII, alpha 1; COMM domain containing 10; COMM domain containing 6; core-binding factor, runt domain, alpha subunit 2; translocated to, 3; cornichon homolog 2 (Drosophila); cortactin; C- terminal binding protein 1 ; C-type lectin domain family 18, member C; CUB and Sushi multiple domains 1; cullin 7; cyclin B2; cyclin Y-like 2; cyclin-dependent kinase 6; cystatin F
  • cysteine statin cysteine sulfinic acid decarboxylase
  • cysteine-rich hydrophobic domain 2 cysteinyl-tRNA synthetase
  • CMP-N-acetylneuraminate monooxygenase pseudogene
  • cytochrome P450 family 4, subfamily F, polypeptide 2
  • cytokine receptor-like factor 3 cytoplasmic FMR1 interacting protein 2
  • DEAD Asp-Glu-Ala-As box polypeptide 19A
  • DEAD/H Asp-Glu-Ala-Asp/His box polypeptide 11 (CHLl-like helicase homolog, S.
  • Drosophila diaphanous homolog 1 (Drosophila); DIP2 disco-interacting protein 2 homolog A (Drosophila); DIS3 mitotic control homolog (S. cerevisiae)-like; discs, large (Drosophila) homolog-associated protein 1; discs, large homolog 1 (Drosophila); discs, large homolog 5 (Drosophila); DnaJ (Hsp40) homolog, subfamily B, member 1; DnaJ (Hsp40) homolog, subfamily C, member 3; DnaJ (Hsp40) homolog, subfamily C, member 7; dopa decarboxylase (aromatic L-amino acid decarboxylase); dopey family member 1 ; dorsal root ganglia homeobox; DOTl-like, histone H3 methyltransferase (S.
  • doublecortin-like kinase 1 doublesex and mab-3 related transcription factor 2; dpy-19-like 1 (C. elegans); dpy-19-like 3 (C. elegans); drebrin-like; dual specificity phosphatase 16; dual specificity phosphatase 6; dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2; dynein, cytoplasmic 1, light intermediate chain 2; dynein, cytoplasmic, light polypeptide pseudogene; dynein, light chain, LC8-type 1; dynein, light chain, LC8-type 2; dysbindin (dystrobrevin binding protein 1) domain containing 1 ; E2F transcription factor 2; early growth response 4; ecotropic viral integration site 5-like; EGF, latrophilin and seven transmembrane domain containing 1 ; egf-like module containing, muc
  • elegans egl nine homolog 2 (C. elegans); ELKS/RAB6-interacting/CAST family member 1; ELKS/RAB6-interacting/CAST family member 1 ; elongation factor Tu GTP binding domain containing 1 ; endoplasmic reticulum-golgi intermediate compartment (ERGIC) 1; endosulfine alpha; engrailed homeobox 1 ; enolase superfamily member 1; epiregulin; epithelial cell transforming sequence 2 oncogene; epithelial membrane protein 1 ; ets variant 6; eukaryotic translation initiation factor IB; eukaryotic translation initiation factor 2 alpha kinase 4; eukaryotic translation initiation factor 3, subunit H; eukaryotic translation initiation factor 5; exocyst complex component 4; exocyst complex component 8; exostoses (multiple)-like 3; extracellular leucine-rich repeat and fibronectin type III
  • GRAM domain containing 1C GRAM domain containing 4; growth differentiation factor 6; growth hormone receptor; GTPase activating protein (SH3 domain) binding protein 1 ; guanidinoacetate N-methyltransferase; guanine nucleotide binding protein (G protein), alpha activating activity polypeptide O; guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 1 ; guanine nucleotide binding protein (G protein), beta polypeptide 1 ; guanine nucleotide binding protein (G protein), gamma 2; guanine nucleotide binding protein (G protein), gamma 7; guanylate binding protein 1, interferon- inducible, 67kDa; HEAT repeat containing 3; HEAT repeat containing 4; heat shock 27kDa protein 2;
  • homeodomain interacting protein kinase 2 homeodomain interacting protein kinase 3; homer homolog 2 (Drosophila); HOP homeobox; HORMA domain containing 2;
  • hyaluronoglucosaminidase 1 hydrocephalus inducing homolog (mouse); hydroxysteroid (17- beta) dehydrogenase 12; hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor); IKAROS family zinc finger 3 (Aiolos); IKBKB interacting protein;
  • interleukin 4 receptor interleukin- 1 receptor-associated kinase 2; internexin neuronal intermediate filament protein, alpha; intracisternal A particle-promoted polypeptide; IQ motif and Sec7 domain 1 ; IQ motif and Sec7 domain 2; IQ motif and Sec7 domain 3; IQ motif containing H; iroquois homeobox 5; isocitrate dehydrogenase 2 (NADP+), mitochondrial; jagged 1 (Alagille syndrome); janus kinase and microtubule interacting protein 2; junctophilin 4; kelch repeat and BTB (POZ) domain containing 7; keratin 222; keratin 73; keratin 82; KH domain containing, RNA binding, signal transduction associated 3; KIAAOlOl ; KIAA0174; KIAA0182; KIAA0240; KIAA0355; KIAA0415; KIAA0913; KIAA
  • Kv channel interacting protein 1 Kv channel interacting protein 2; 1(3 )mbt- like 4 (Drosophila); lactamase, beta 2; lactate dehydrogenase D; laminin, alpha 1 ; latrophilin 1 ; Leber congenital amaurosis 5; lectin, galactoside-binding, soluble, 14; lemur tyrosine kinase 3; Leol, Pafl/RNA polymerase II complex component, homolog (S.
  • leucine aminopeptidase 3 leucine carboxyl methyltransferase 1 ; leucine rich repeat and fibronectin type III domain containing 5; leucine rich repeat and Ig domain containing 1 ; leucine rich repeat and sterile alpha motif containing 1 ; leucine rich repeat containing 37, member A2; leucine rich repeat containing 37, member A4 (pseudogene); leucine rich repeat containing 37A; leucine rich repeat containing 61 ; leucine rich repeat containing 68; leucine rich repeat containing 8 family, member A; leucine- rich repeat kinase 1 ; leucine-rich repeats and calponin homology (CH) domain containing 3; leucine-rich repeats and immunoglobulin-like domains 2; leukocyte cell derived chemotaxin 1; LIM domain and actin binding 1; LIM domain binding 2; LIM domain only 1 (rhombotin 1); LIM home
  • lipase maturation factor 1 lipase, hepatic; lipocalin 8; low density lipoprotein receptor class A domain containing 1 ; low density lipoprotein receptor-related protein 4; lymphocyte antigen 6 complex, locus E; lymphocyte antigen 9; LYR motif containing 4; lysophospholipase I; lysosomal protein transmembrane 4 beta; lysyl oxidase-like 1; mahogunin, ring finger 1; major facilitator superfamily domain containing 1 1; major histocompatibility complex, class I, A; major histocompatibility complex, class II, DM alpha; major histocompatibility complex, class II, DM beta; major vault protein; makorin ring finger protein 2; mannosyl (beta- 1 ,4-)-glycoprotein beta-l,4-N- acetylglucosaminyltransferase; MAP7 domain containing 2; mastermind-
  • metastasis associated 1 metastasis associated 1 family, member 3; metastasis associated in colon cancer 1 ; methionine adenosyltransferase II, beta; methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2-like; methyltransferase like 14; methyltransferase like 2B;
  • myeloid/lymphoid or mixed-lineage leukemia trithorax homolog, Drosophila; translocated to, 1; myeloid/lymphoid or mixed-lineage leukemia 2; myosin ID; myosin VB; myosin VC; myosin VIIA and Rab interacting protein; myosin VIIB; myosin, heavy chain 3, skeletal muscle, embryonic; myosin, light chain 12A, regulatory, non-sarcomeric; myosin, light chain 4, alkali; atrial, embryonic; myozenin 3; NACC family member 2, BEN and BTB (POZ) domain containing; N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase); N- acetyltransferase 1 (arylamine N-acetyltransferase); NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4,
  • neurocanthocytosis neurofibromin 1 ; neurogenic differentiation 6; neuroligin 4, X-linked;
  • nucleobindin 2 nucleolar protein 10; nucleoporin 107kDa; nucleoporin 188kDa; nucleoporin 214kDa; NudC domain containing 1 ; nudix (nucleoside diphosphate linked moiety X)-type motif 13; numb homolog (Drosophila); nurim (nuclear envelope membrane protein); olfactomedin-like 1; olfactory receptor, family 1, subfamily J, member 1 ; olfactory receptor, family 11, subfamily H, member 2; olfactory receptor, family 4, subfamily F, member 29; olfactory receptor, family 7, subfamily E, member 8 pseudogene; oncomodulin 2; Opa interacting protein 5; Opa interacting protein 5; orofacial cleft 1 candidate 1; otopetrin 1 ; OTU domain containing 7A; PAN3 poly(A) specific ribonuclease subunit homolog (S. cerevisi
  • PBX/knotted 1 homeobox 1 PCF11, cleavage and polyadenylation factor subunit, homolog (S. cerevisiae); PDS5, regulator of cohesion maintenance, homolog A (S. cerevisiae); PDZ and LIM domain 2 (mystique); PDZ and LIM domain 7 (enigma); PDZ domain containing ring finger 3; peptidylprolyl isomerase A (cyclophilin A)-like 4A; peripherin;
  • peroxiredoxin 6 peroxisome proliferator-activated receptor gamma, coactivator 1 beta; PH domain and leucine rich repeat protein phosphatase 1 ; PHD and ring finger domains 1 ; PHD finger protein 12; PHD finger protein 13; PHD finger protein 17; phosphatase and tensin homolog; phosphatidic acid phosphatase type 2B; phosphatidylethanolamine-binding protein 4; phosphatidylinositol binding clathrin assembly protein; phosphatidylinositol glycan anchor biosynthesis, class B; phosphatidylinositol transfer protein, membrane-associated 2;
  • phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 phosphatidylinositol- 4-phosphate 5-kinase, type I, beta; phosphodiesterase 3A, cGMP-inhibited; phosphoenolpyruvate carboxykinase 1 (soluble); phosphoglucomutase 2-like 1 ; phosphoglucomutase 5;
  • phosphoinositide-3-kinase class 2, alpha polypeptide
  • phosphoinositide-3-kinase class 3
  • phospholipase A2 group IVC (cytosolic, calcium-independent); phospholipase A2, group IVE; phosphoprotein associated with glycosphingolipid microdomains 1 ; phosphorylase kinase, alpha
  • pleomorphic adenoma gene-like 1 pleomorphic adenoma gene-like 1 ; plexin CI ; PMS1 postmeiotic segregation increased 1 (S. cerevisiae); PNMA-like 1 ; poliovirus receptor-related 1 (herpesvirus entry mediator C); polo-like kinase 1 (Drosophila); poly(A) binding protein, cytoplasmic 4 (inducible form); poly(rC) binding protein 1; polybromo 1 ; polycystic kidney disease (polycystin) and REJ homolog (sperm receptor for egg jelly homolog, sea urchin); polymerase (RNA) I polypeptide A, 194kDa; polymerase (RNA) III (DNA directed) polypeptide B; post-GPI attachment to proteins 2;
  • potassium channel tetramerisation domain containing 21 potassium channel tetramerisation domain containing 7; potassium channel, subfamily T, member 2; potassium inwardly-rectifying channel, subfamily J, member 16; potassium voltage-gated channel, delayed-rectifier, subfamily S, member 1 ; potassium voltage-gated channel, KQT-like subfamily, member 2; potassium voltage-gated channel, Shaw-related subfamily, member 2; PPPDE peptidase domain containing 2; PR domain containing 11 ; PR domain containing 14; PR domain containing 4; prickle homolog 1 (Drosophila); primase, DNA, polypeptide 2 (58kDa); programmed cell death 7; prolactin; proline rich 14; proline rich 7 (synaptic); prolyl 4-hydroxylase, alpha polypeptide I; proprotein convertase subtilisin/kexin type 6; prostate stem cell antigen; prostate tumor overexpressed 1 ; protease, serine, 12 (neurotrypsin,
  • pseudouridylate synthase 10 PTPRF interacting protein, binding protein 1 (liprin beta 1);
  • purinergic receptor P2X ligand-gated ion channel, 4; purine-rich element binding protein A; pyrroline-5-carboxylate reductase 1 ; quaking homolog, KH domain RNA binding (mouse); quiescin Q6 sulfhydryl oxidase 1 ; RAB GTPase activating protein 1; RAB27B, member RAS oncogene family; RAB2A, member RAS oncogene family; RAB3A interacting protein (rabin3); RAB6C, member RAS oncogene family; RAB7, member RAS oncogene family-like 1; rabaptin, RAB GTPase binding effector protein 1 ; RADl homolog (S.
  • pombe RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae); RAD52 motif 1 ; Rap guanine nucleotide exchange factor (GEF) 3; RAPl interacting factor homolog (yeast); RAR-related orphan receptor A; Ras association (RalGDS/AF-6) domain family (N-terminal) member 10; Ras association
  • regulatory factor X 3 (influences HLA class II expression); reticulocalbin 1, EF-hand calcium binding domain; reticulon 2; reticulon 4; retinoic acid receptor, alpha; retinol dehydrogenase 13 (all-trans/9-cis); retrotransposon gag domain containing 4; REX1, RNA exonuclease 1 homolog (S. cerevisiae); REXl, RNA exonuclease 1 homolog (S. cerevisiae)-like 2 (pseudogene);
  • Rho GTPase activating protein 1 1A Rho GTPase activating protein 1 IB; Rho GTPase activating protein 22; Rho guanine nucleotide exchange factor (GEF) 10-like; rhomboid 5 homolog 2 (Drosophila); rhophilin, Rho GTPase binding protein 1; Rho-related BTB domain containing 2; rhotekin 2; Rhox homeobox family, member 2; ribosomal LI domain containing 1 ; ribosomal L24 domain containing 1 ; ribosomal protein L30; ribosomal protein S7; ribosomal protein SA pseudogene 58; ribosomal protein, large, PI ; ring finger protein 139; ring finger protein 145; ring finger protein 152; ring finger protein 157; ring finger protein 165; ring finger protein 220; ring finger protein 24; ring finger finger protein 24; ring finger finger
  • pombe roundabout, axon guidance receptor, homolog 3 (Drosophila); RPGRIP 1 -like; RRS1 ribosome biogenesis regulator homolog (S. cerevisiae); RUN and FYVE domain containing 1; RUN and FYVE domain containing 3; SI 00 calcium binding protein A2; saccharopine dehydrogenase (putative); sal-like 3 (Drosophila); SAM and SH3 domain containing 1; SAP30-like; sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein); sarcosine dehydrogenase; SATB homeobox 1; scaffold attachment factor B2; schlafen family member 5; scinderin; SCYl-like 1 (S. cerevisiae); Sec23 homolog B (S.
  • secretagogin EF-hand calcium binding protein
  • secreted protein acidic, cysteine- rich (osteonectin); secretory carrier membrane protein 5; sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B; septin 9; serine hydroxymethyltransferase 1 (soluble); serine racemase;
  • serine/arginine repetitive matrix 3 serine/threonine kinase 17b; serine/threonine kinase 24 (STE20 homolog, yeast); serpin peptidase inhibitor, clade B (ovalbumin), member 9; SERPINEl mRNA binding protein 1 ; sestrin 3; SET and MYND domain containing 3; SET binding factor 2; SET binding protein 1 ; SET domain containing 6; seven in absentia homolog 1 (Drosophila); sex comb on midleg homolog 1 (Drosophila); SH3 and PX domains 2A; SH3 domain containing 19; SH3 -binding domain kinase 1; SHC (Src homology 2 domain containing) family, member 4; Shwachman-Bodian-Diamond syndrome; sialidase 3 (membrane sialidase); sideroflexin 5; signal peptide, CUB domain, EGF-like 3; signal recognition particle 54kDa; signal-
  • SIK family kinase 3 sine oculis binding protein homolog (Drosophila); single-stranded DNA binding protein 2; SMAD family member 3; SMAD family member 6; small EDRK-rich factor 1A (telomeric); SMEK homolog 1, suppressor of mekl (Dictyostelium); Smg-5 homolog, nonsense mediated mRNA decay factor (C. elegans);
  • solute carrier family 14 urea transporter
  • member 2 solute carrier family 16, member 3 (monocarboxylic acid transporter 4); solute carrier family 16, member 4 (monocarboxylic acid transporter 5); solute carrier family 2 (facilitated glucose transporter), member 11 ; solute carrier family 22, member 23; solute carrier family 25 (mitochondrial carrier; peroxisomal membrane protein, 34kDa), member 17; solute carrier family 25, member 13 (citrin); solute carrier family 25, member 30; solute carrier family 26, member 10; solute carrier family 35, member F5; solute carrier family 37 (glycerol-3- phosphate transporter), member 3; solute carrier family 39 (metal ion transporter), member 11 ; solute carrier family 39 (zinc transporter), member 14; solute carrier family 39 (zinc transporter), member 2; solute carrier family 46, member 2; solute carrier family 6 (neurotransmitter transporter, taurine
  • tetratricopeptide repeat domain 30A tetratricopeptide repeat domain 7B; tetratricopeptide repeat domain 8; tetratricopeptide repeat domain 9; thioredoxin-related transmembrane protein 3;
  • thrombospondin 2 thrombospondin 4; thymopoietin; thyroid hormone receptor, beta
  • transcription factor 12 transcription factor 12; transcription factor 4; transcription factor 7-like 1 (T-cell specific, HMG- box); transcription factor AP-4 (activating enhancer binding protein 4); transcription factor Dp-1; transcription factor Dp-2 (E2F dimerization partner 2); transducin (beta)-like 1 X-linked receptor 1; transducin-like enhancer of split 3 (E(spl) homolog, Drosophila); transferrin receptor (p90, CD71); transient receptor potential cation channel, subfamily A, member 1 ; transmembrane 6 superfamily member 2; transmembrane 7 superfamily member 3; transmembrane 9 superfamily member 2; transmembrane and coiled-coil domain family 1; transmembrane and coiled-coil domains 4; transmembrane and ubiquitin-like domain containing 2; transmembrane emp24 domain trafficking protein 2; transmembrane protein 131 ; transmembrane protein 132C;
  • transmembrane protein 132D transmembrane protein 135; transmembrane protein 14B;
  • transmembrane protein 164 transmembrane protein 17; transmembrane protein 180;
  • transmembrane protein 181 transmembrane protein 181; transmembrane protein 183A; transmembrane protein 188;
  • transmembrane protein 219 transmembrane protein 233; transmembrane protein 45B;
  • transmembrane protein 59 transmembrane protein 64; transmembrane protein 66;
  • transmembrane protein 66 transmembrane protein 66; transmembrane protein 86B; transthyretin; trichohyalin-like 1 ;
  • trophinin associated protein tastin
  • tropomyosin 3 tubby homolog (mouse); tuberous sclerosis 1 ; tubulin, beta 2B; tumor necrosis factor receptor superfamily, member 10c, decoy without an intracellular domain; tumor necrosis factor receptor superfamily, member 1A; tweety homolog 2 (Drosophila); tyrosylprotein sulfotransferase 1 ; ubiquinol-cytochrome c reductase, Rieske iron- sulfur polypeptide 1; ubiquitin C; ubiquitin protein ligase E3 component n-recognin 3 (putative); ubiquitin protein ligase E3 component n-recognin 5; ubiquitin specific peptidase 10; ubiquitin specific peptidase 15; ubiquitin specific peptidase 29; ubiquitin specific peptidase 8; ubiquitin- conjugating enzyme E2D
  • elegans uveal autoantigen with coiled-coil domains and ankyrin repeats
  • vaccinia related kinase 2 vaccinia related kinase 3
  • vacuolar protein sorting 13 homolog A S. cerevisiae
  • vacuolar protein sorting 24 homolog S. cerevisiae); vestigial like 4 (Drosophila); vinculin; vitamin K epoxide reductase complex, subunit 1 -like 1 ; v-myc myelocytomatosis viral oncogene homolog (avian); Vpr (HIV-1) binding protein; v-ral simian leukemia viral oncogene homolog B (ras related; GTP binding protein); v-ski sarcoma viral oncogene homolog (avian); WAS protein family homolog 2 pseudogene; WD repeat domain 1 ; WD repeat domain 20; WD repeat domain 27; WD repeat domain 31 ; WD repeat domain 44; WD repeat domain 70; WD repeat domain 85; Werner helicase interacting protein 1; Williams-Beuren syndrome chromosome region 16;
  • wingless-type MMTV integration site family member 3 A; WNT1 inducible signaling pathway protein 3 ; WW domain binding protein 1 1 ; WWC family member 3 ; X-linked inhibitor of apoptosis; XRCC6 binding protein 1; YTH domain family, member 1 ; zinc finger and AT hook domain containing; zinc finger and BTB domain containing 45; zinc finger and BTB domain containing 9; zinc finger and SCAN domain containing 22; zinc finger CCCH-type, antiviral 1; zinc finger E-box binding homeobox 1 ; zinc finger protein 100; zinc finger protein 114; zinc finger protein 121; zinc finger protein 160; zinc finger protein 161 homolog (mouse); zinc finger protein 19; zinc finger protein 192; zinc finger protein 205; zinc finger protein 213; zinc finger protein 254; zinc finger protein 256; zinc finger protein 263; zinc finger protein 283; zinc finger protein 30; zinc finger protein 318; zinc finger protein 322B; zinc finger protein 329; zinc finger protein 354B; zinc finger protein
  • IncRNA function or levels can be used to regulate chromatin status, gene expression, transcription, translation, post-translational events and global biomolecular trafficking in the cell, especially to and from the nucleus.
  • Methods of designing, modulating or targeting IncRNAs may be either structure-based or sequence based. Traditionally, methods of targeting nucleic acid molecules in the cell have been sequenced based and have depended in some form on harnessing the hybridization or base pairing of two complementary molecules. Sequence-based methods of modulating or altering IncRNA function and levels are described herein.
  • structure based methods are those methods of altering or modulating a IncRNA function or level that depends on the determination or knowledge of the higher order structure of at least a portion of a IncRNA target.
  • “Higher order structures” include but are not limited to the overall secondary, tertiary or quarternary structure of a molecule, e.g., hairpin structures, bulges, etc. These structures may be determined informatically with prediction algorithms based on thermodynamic parameters and energy calculations. Preferably, secondary structure prediction is performed using either M-fold or RNA Structure algorithm. Programs for secondary structure determination are freely available online. Structures may also be determined by NMR, Mass Spectroscopy or by crystallographic methods.
  • RNA molecules methods of determining overall structure or structures of portions of the RNA molecule are known in the art.
  • methods of identifying secondary structures in eukaryotic and prokaryotic RNA molecules termed "molecular interaction sites.”
  • Molecular interaction sites are small, usually less than 30 nucleotides, independently folded, functional subdomains contained within a larger RNA molecule. These methods may be used to determine molecular interaction sites on IncRNAs.
  • IncRNA targets may also be subjected to mimicry design. Disclosed in US Patent 6,368,863 incorporated herein by reference in its entirety, are methods of identifying protein interacting sites on an RNA molecule and then designing an oligonucleotide that mimics that portion of the larger RNA molecules. These methods may be used in the present invention to design small IncRNA target mimics which will bind proteins.
  • structure-based targeting Unlike sequence-based or hybridization driven targeting, which must rely on access of the targeting molecule to the target in order for base pairing to occur, structure-based targeting embraces a larger portion of the IncRNA target.
  • IncRNAs are defined as distinct nucleic acid-based components of the molecule.
  • Features of the IncRNAs of the present invention may be structural features and may include surface manifestations, local conformational shape, folds, loops, half- loops, domains, half-domains, sites, termini or any combination thereof.
  • the starting molecule may be one selected from Table 2 or known in the cell as the wild type molecule. Alternatively, a series of modifications may be made in which the starting molecule may be referred to simply as the parent molecule.
  • Structural features of the present invention may be at least 200 nucleotides in length or from about 200 to about 500 nucleotides in length or from about 200 to about 300 nucleotides in length or from about 50 to about 100 nucleotides in length. They may also comprise the whole or any part of a defined structural feature. Structural features may be 4-10, 5-15, 10-20, 10-30, or 20-50 nucleotides in length. These may be represented in increments of the triplet code and therefore may be any multiple of three. For example, features may be from 15-18, 15-30, 15-36, 15-60, 30-60, 30-90, 30-120 or larger.
  • the term "surface manifestation" refers to a nucleic acid based component of a IncRNA appearing on an outermost surface of the IncRNA.
  • the term "local conformational shape" means a nucleic acid based structural manifestation of a IncRNA which is located within a definable space of the IncRNA.
  • fold means the resultant conformation of a nucleic acid sequence upon energy minimization.
  • a fold may occur at the secondary or tertiary level of the folding process. Examples of secondary level folds include hairpins, loops and bulges. Examples of tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include
  • hydrophobic and hydrophilic pockets and the like.
  • the term "turn” as it relates to IncRNA conformation means a bend which alters the direction of the backbone of a poly- or oligonucleotide and may involve one, two, three or more nucleotides.
  • loop refers to a structural feature of a poly- or oligonucleotide which reverses the direction of the backbone of a poly- or oligonucleotide and comprises four or more nucleotides.
  • domain refers to a motif of a poly- or oligonucleotide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions, etc).
  • half-domain means portion of an identified domain having at least half the number of nucleotides as the domain from which it is derived. It is understood that domains may not always contain an even number of nucleotides. Therefore, in those cases where a domain contains or is identified to comprise an odd number of nucleotides, a half-domain of the odd-numbered domain will comprise the whole number portion or next whole number portion of the domain (number of nucleotides of the domain/2+/-0.5 nucleotides).
  • sub-domains may be identified within domains or half- domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the nucleotides that comprise any of the domain types herein need not be contiguous along the backbone of the poly- or oligonucleotide (i.e., nonadjacent nucleotides may fold structurally to produce a domain, half-domain or subdomain).
  • site represents a location for targeting a IncRNA.
  • a site represents a position within a poly- or oligonucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention.
  • sites of targeting can represent hundreds to thousands of nucleotides and may include nucleotides very distal in sequence location.
  • IncRNAs may present surfaces, domains or sites which comprise nucleotides which ony appear juxtaposed due to the folded nature of the IncRNA.
  • LDAs may target any site on a IncRNA.
  • terminal or terminus when referring to IncRNAs refers to an extremity of a poly- or oligonucleotide. Such extremity is not limited only to the first or final site of the poly- or oligonucleotide but may include additional nucleotides in the terminal regions.
  • the poly- or oligonucleotide based molecules of the present invention may be characterized as having both a 5' and a 3 ' terminus.
  • Poly- or oligonucleotides of the invention are in some cases made up of multiple chains brought together by disulfide bonds or by non- covalent forces (multimers, oligomers or dendrimers).
  • poly- or oligonucleotides will have multiple 5' and 3 '-termini.
  • termini of the poly- or oligonucleotide may be modified such that they begin or end, as the case may be, with a non- poly- or oligonucleotide based moiety such as a conjugate.
  • any of the features have been identified or defined as a component of a IncRNA of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating them to produce additional IncRNA variants.
  • a IncRNA transcript variant is designed to encode a LDA.
  • a IncRNA transcript RNA molecule may comprise the sequence of a shRNA or other nucleic acid based LDA.
  • the LDA nucleic acid based
  • the LDA may be one that targets a different site on the IncRNA in which is is encoded or it may target the RNA transcript of a coding gene or any nucleic acid based transcript to which it will either hybridize (sequence based targeting) or form an interation with (structure based targeting).
  • Modifications and manipulations can be accomplished by methods known in the art.
  • the resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
  • a feature of a IncRNA transcript is removed to produce a IncRNA transcript variant.
  • a feature of a IncRNA transcript is duplicated to produce a IncRNA transcript variant.
  • a feature of a IncRNA is swapped with a second feature of a IncRNA to produce a IncRNA variant.
  • the second feature is from the same or a different IncRNA transcript.
  • hairpin features of a IncRNA are altered or modified. Hairpin structures of a first IncRNA may be inserted into a second IncRNA. They may also be removed from a IncRNA. Where a feature is found to have a biological activity such as an interface for binding, or as a signal for localization, the feature may be reproduced in isolation by chemical or synthethic methods and used as a IncRNA directed agent of the invention.
  • IncRNAs may be targeted to alter cellular memory, or cell identity. Without wishing to be bound by theory, it is believed that the IncRNAs may contribute to cellular memory and play a determinative role in the cells ability to produce daughter cells of the same lineage or RNA population signature thereby maintaining the identity of the cells during cell divisions.
  • the "RNA population signature" of a cell is the qualitative complement of RNA transcripts present in a cell at a particular time or timeframe that distinguishes the cell from other cell types. It should be understood that an RNA population signature of a cell does not necessarily comprise the sum total of all RNAs present in a cell but a set or subset of transcripts which may be used to identify one cell type from another cell type.
  • RNA population signatures may comprise "nuclear signatures", “cytoplasmic signatures", “organelle associated signatures,” “tissue-associated signatures,” or combinations thereof. They may also comprise the set of IncRNA genes or transcripts or subsets thereof.
  • the RNA nuclear population signature of a cell may comprise the set or subset of RNA transcripts present in the nucleus of the cell at a particular time or developmental phase such that this signature can be compared to other cells in order to determine whether the cells are of the same type or along the same path of lineage.
  • Methods of measuring the presence of RNA in a cell are well known in the art.
  • Methods of determining the cell type of a specific cell include for example the measurement of cell type specific markers such as proteins or protein or ligand expression, receptor or ligand presence or secretion.
  • the RNA population signature of a cell may be measured or identified and compared to another cell. The comparison will reveal differences between the two signatures. Differences in the IncRNA transcript components of the signature may then be assessed and a cellular RNA population may be supplemented or reduced to effect a similar signature in the target cell population.
  • Cells of the present invention include, but are not limited to, cells that are derived primarily from endoderm (gland cells, exocrine secretory epithelial cells, hormone secreting cells, epithelial cells lining closed internal body cavities); cells derived primarily from ectoderm (integumentary system, keratinizing epithelial cells, wet stratified barrier epithelial cells, nervous system cells, sensory transducer cells, autonomic neuron cells, sense organ and peripheral neuron supporting cells, central nervous system neurons and glial cells, lens cells); cells derived primarily from mesoderm (metabolism and storage cells, barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract), kidney; extracellular matrix secretion cells, contractile cells, blood and immune system cells, pigment cells, nurse cells and interstitial cells).
  • Stem cells include, but are not limited to adult, embryonic, pluripotent, totipotent, and induced pluripotent.
  • RNA population signature may be altered by adding back one or more IncRNA genes or transcripts.
  • cellular processes are altered such that the cell may differentiate along a different path to alter the phenotype of the cell or to mirror the RNA population signature of a target cell.
  • RNA population signature of a cell may be altered by administering an LDA or an exogenously prepared IncRNA gene or transcript, the outcomes of which would result in the alteration of the cellular phenotype (See Arancio, W., Rejuvenation Research, 13, 1-8, 2010).
  • cells may be treated to alter the lineage or differentiation state of for example stem cells or cells of early developmental lineage.
  • the RNA population signature of a cell, cell line or tissue may be used in diagnostic applications.
  • methods are provided for the use of the RNA population signature, more specifically the IncRNA population signature of a cell or tissue, in diagnostic applications.
  • IncRNAs associated with a disease or condition or having a linkage related to chromosome location are measured and where the level of IncRNAs are not similar or identical to a normal cell of the same lineage, one or more LDAs are administered which increase or decrease the level of IncRNA as desired.
  • the milieu of a cell or tissue may be used to provide a signaling environment for the study of IncRNA regulation.
  • the term "milieu of a cell or tissue” means the supernatant or "soup" of a cell population or an extract of the cell system.
  • a first population of cells may be incubated in the milieu of a second population of cells in order to provide an environment which alters the expression levels or RNA population signature of the first population of cells.
  • To the milieu may be added one or more IncRNA genes, transcripts or LDAs of the present invention.
  • IncRNA genes, transcripts or LDAs of the present invention As a consequence of this addition the development, differentiation or overall gene expression profile of the first population of cells may be changed.
  • Cells which may be used to provide the incubating milieu or which may be incubated in the milieu include but are not limited to somatic or gamete, stem cells, pluripotetent cells, cells of primary origin, cells of any mammalian tissue, etc.
  • chromatin inactivation or activation may be effected by the administration of one or more LDAs (whether sequence based or structure based) or the administration of one or more IncRNA transcripts.
  • LDAs whether sequence based or structure based
  • IncRNA transcripts may be added to cells or cell systems to alter the epigenetic landscape of a cell or tissue.
  • IncRNA cassettes may be added to or administered to a cell or tissue.
  • a "IncRNA cassette” is a polynucleotide that encodes one or more IncRNAs.
  • IncRNA cassettes may endcode a full length wild type IncRNA or may be designed to encode a modified IncRNA.
  • modified IncRNA means a IncRNA which differs from the wild type sequence of the IncRNA in question. Modifications to IncRNAs include those modifications to the exonic structure of the IncRNA and include those having shuffled exon structures, omitted exons and additional exons.
  • a wild type IncRNA will never encode a mature protein (greater than 50 amino acids), however it is contemplated by the present invention that a IncRNA or IncRNA cassette may be designed which contains one or more complete or partial exons from a coding gene transcript.
  • modified IncRNA transcripts could be capable of encoding a peptide or polypeptide sequence.
  • the IncRNA cassette may also contain a modified promoter from that which is found in the wild type IncRNA. Promoters may be swapped with those of protein coding genes or other IncRNA genes. They may also be modified by addition, deletion or shuffling of promoter components. Synthetic IncRNA genes or transcripts may be modified before contacting or administration to cells and the promoters may have temporary or permanent tags or transcription factors pre-associated with them. LDAs of the present invention may also be designed to target the promoters of IncRNA genes. In this design, the LDAs may be linked to, conjutaged, associated or complexed with factors that target the LDA to the site of the IncRNA in the cell.
  • IncRNA transcripts do not encode mature proteins. However, it is contemplated that IncRNA transcripts may have the capacity to encode smaller proteins or peptides (Kondo, et al, Science 16 July 2010: Vol. 329. no. 5989, pp. 336 - 339).
  • peptide is an amino acid based molecule of no more than 50 amino acids.
  • IncRNA transcripts are found to encode or engineered to encode peptides, these peptides or their locus within the IncRNA transcript may be a target of the invention. Consequently, methods of regulating IncRNA transcripts of the present invention may also regulate their encoded peptides.
  • synthetic refers to a state of having been created or man-made, e.g., not of natural origin.
  • the LDAs or IncRNAs of the present invention may be synthesized using chemical or enzymatic or recombinant methods. They may then be isolated from the synthetic mixture. Compounds of the present invention may also be isolated from a natural source.
  • LDAs or IncRNAs of the present invention may be associated with chromatin modifying complexes, nucleosome components, proteins or enzymes. They may also be modified to localize to either the nucleus or cytoplasm of the cells. In this manner, the LDAs or IncRNAs of the present invention may be guided to specific sites in a cell or tissue and may affect cellular processes such as gene expression, imprinting, aging, epigenetic signatures and the like.
  • the invention provides a method for modulating (e.g., inhibiting or activating) the expression of a IncRNA in a mammal.
  • ncRNAs especially IncRNAs include those described in WO/2005/060344 and WO/2003/025229 (describing efference RNA or eRNA) the contents of which are incorporated herein by reference in its entirety.
  • LDAs IncRNA Directed Agents
  • compositions of the present invention are those which may be used to regulate, control, manipulate, perturb or otherwise alter the expression, levels, activity or status of IncRNAs.
  • compositions of the present invention are termed "IncRNA-directed agents" or "LDAs”.
  • the LDAs of the present invention broadly include, but are not limited to, oligonucleotides, polynucleotides, iRNA agents, antisense molecules, ribozymes, aptamers, small molecules, antibodies, peptides, proteins, enzymes or fragments thereof, and vitamins.
  • the method includes administering an LDA composition featured in the invention to the mammal such that expression of the target IncRNA is decreased, such as for an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer.
  • an extended duration e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer.
  • the method includes administering a composition as described herein to a mammal such that expression of the target IncRNA is increased by e.g., at least 10% compared to an untreated animal.
  • the activation of IncRNA occurs over an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, four weeks, or more.
  • a LDA can activate IncRNA expression by stabilizing the IncRNA transcript, interacting with a promoter in the genome, and/or inhibiting an inhibitor of IncRNA expression.
  • the LDAs useful for the methods and compositions featured in the invention specifically target RNAs (primary or processed) of the target IncRNA.
  • Compositions and methods for inhibiting the expression of these IncRNAs using iRNAs can be prepared and performed as described elsewhere herein.
  • the method includes administering a composition containing a LDA, where the LDA includes a nucleotide sequence that is complementary to at least a part of an RNA or DNA transcript of the IncRNA of the mammal to be treated.
  • Mammals include, but are not limited to, humans, monkeys, rodents, rabbits, dogs, cats, pigs, cows, horses and the like.
  • compositions and methods of the present invention find utility in research, discovery, diagnostics and therapeutic areas of human medicine, veterinary medicine, plant science and the control of pests, insects and the like.
  • the composition may be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration.
  • intracranial e.g., intraventricular, intraparenchymal and intrathecal
  • intravenous intramuscular
  • subcutaneous e.g., transdermal
  • transdermal e.g., transdermal
  • airway e.g., aerosol
  • nasal e.g., fenchymal and intrathecal
  • topical including buccal and sublingual
  • the compositions are administered by intravenous infusion or injection.
  • LDAs may be designed to target regions or sites along a IncRNA which correlate to hypersensitivity sites (HS) found on the corresponding DNA encoding the IncRNA.
  • HS hypersensitivity sites
  • Method of identification are described in PCT Publication WO/2004/053106, the contents of which are incorporated by reference herein in its entirety.
  • an LDA as described herein effects inhibition IncRNA
  • an LDA as described herein activates IncRNA expression.
  • the term “modulate the expression of,” refers to an at least partial “inhibition” or partial “activation” of a IncRNA gene or transcript expression in a cell treated with a LDA composition as described herein compared to the expression of a IncRNA gene or transcript in an untreated cell. Modulation of expression may be determined not only by direct measurement of a IncRNA level after contacting with the LDA, but also be inference by associating a known phenotypic outcome which correlates to said contacting.
  • the LDAs of the present invention may target, mimic, bind to, replace or alter the levels or function of a product of a IncRNA.
  • IncRNA products include any molecule engineered to be a product of a IncRNA either by transcription, translation, cleavage, splicing, or other mechanism that produces a derivative of a IncRNA.
  • Examples of IncRNA products include, but are not limited to, peptides or proteins engineered to be coded by the IncRNA or fragments of the IncRNA transcript.
  • expression of a IncRNA transcript is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a LDA as described herein.
  • a IncRNA is activated by at least about 60%, 70%, or 80% by administration of a LDA featured in the invention.
  • expression of a IncRNA transcript is activated by at least about 85%, 90%, or 95% or more by administration of a LDA as described herein.
  • the IncRNA transcript expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with a LDA as described herein compared to the expression in an untreated cell.
  • Activation of expression of coding mRNAs by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Set U.S.A. 103 : 17337-42, and in US200701 11963 and US2005226848, each of which is incorporated herein by reference. It is believed that constructs that activate the expression of coding RNA transcripts will also activate the expression of non-coding RNA transcripts such as the lncRNA transcripts of the present invention.
  • the degree of inhibition is usually expressed in terms of:
  • inhibition or the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to lncRNA expression, e.g., the amount of protein encoded by a mRNA that itself is controlled by a lncRNA, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production or the status of a cell, e.g., the epigenetic profile or signature of a cell which is altered upon modulation of one or more lncRNA targets.
  • a parameter that is functionally linked to lncRNA expression e.g., the amount of protein encoded by a mRNA that itself is controlled by a lncRNA
  • the number of cells displaying a certain phenotype e.g., lack of or decreased cytokine production or the status of a cell, e.g., the epigenetic profile or signature of a cell which is altered upon modulation of one or more lncRNA targets.
  • lncRNA silencing may be determined in any cell expressing lncRNA, either constitutively or by genomic engineering, and by any appropriate assay.
  • expression of a lncRNA is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a LDA featured in the invention.
  • a lncRNA is suppressed by at least about 60%, 70%, or 80% by administration of a LDA featured in the invention.
  • a lncRNA is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of a LDA as described herein.
  • RNA agents include, but are not limited to, small interfering RNAs (siRNA), double stranded RNAs (dsRNAs), inverted repeats, short hairpin R As (shRNAs), small temporally regulated RNAs (stR A), clustered inhibitory RNAs (cRNAs), including radial clustered inhibitory RNA, asymmetric clustered inhibitory RNA, linear clustered inhibitory RNA, and complex or compound clustered inhibitory RNA, dicer substrates, DNA-directed RNAi (ddRNAi), single-stranded R Ai (ssRNAi), microRNA (miRNA) antagonists, microRNA mimics, microRNA agonists, blockmirs (a.k.a.
  • RNAi constructs such as those disclosed in US Publication 20090131360, the contents of which are incorporated herein in its entirety, the solo-rxRNA constructs disclosed in PCT Publication WO/2010/01 1346, the contents of which are incorporated herein by reference in its entirety; the sd-rxRNA constructs disclosed in PCT Publication WO/2010/033247 the contents of which are incorporated herein by reference in its entirety, dual acting RNAi constructs which reduce RNA levels and also modulate the immune response as disclosed in PCT Publications WO/2010/002851 and WO/2009/141146 the contents of which are incorporated herein by reference in their entirety and antigene RNAs (agRNA) or small activiating RNAs (saRNAs) which increase expression of
  • the term "iRNA” refers to an agent that comprises at least an oligonucleotide component (e.g., nucleic acid, either RNA or DNA or modifications thereof), and which is capable of functioning through binding, preferably via hybridization.
  • the iRNA agent mediates the targeted cleavage of an RNA transcript via an RNA- induced silencing complex (RISC) pathway.
  • RISC RNA- induced silencing complex
  • an iRNA agent as described herein effects inhibition IncRNA expression.
  • a iRNA agent as described herein activates IncRNA expression.
  • an iRNA agent sterically blocks access to at least a portion of the IncRNA target. Such blocking can result in the modulation of IncRNA expression, levels or function.
  • iRNA agents may act via binding but not trigger any cleavage event, but exert an effect on the function of the IncRNA target by steric means.
  • the agent may block the site of another moiety which normally would bind to the IncRNA to itself effect cleavage.
  • the iRNA agent will comprise nucleic acid and non-nucleic acid components and the nucleic acid component may be responsible for the binding but not directly for the alteration in function of the IncRNA target.
  • conjugates of iRNA agents may have two or more functions with the nucleic acid component providing at least the hybridization function, while second, third or additional components provide functional effect to the LDA.
  • target sequence refers to a contiguous portion of the nucleotide sequence of a DNA molecule of a IncRNA gene or RNA sequence formed during the transcription of a IncRNA, including the IncRNA transcript that is a product of RNA processing of a primary transcription product.
  • the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed activity/ e.g., cleavage, blocking, etc) at or near that portion.
  • the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween.
  • the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides,20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides,
  • target sequence can be from 9-15 nucleotides, 10-12 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides.
  • strand comprising a sequence refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
  • the term "complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person.
  • Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing.
  • stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing.
  • Other conditions such as physiologically relevant conditions as may be encountered inside an organism, or cell or tissue or cell culture can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
  • Complementary sequences within a LDA include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over a portion of one or both nucleotide sequences.
  • base-pairing is over the entire length of both sequences, such sequences can be referred to as "fully complementary" with respect to each other herein.
  • first sequence is referred to as “substantially complementary” with respect to a second sequence herein
  • the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., modulation of gene expression via a R Ai pathway.
  • mismatches may be as many as 10, 20, 30 or more up to 25% of the molecule.
  • a dsR A comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter
  • oligonucleotide may yet be referred to as "fully complementary" for the purposes described herein.
  • “Complementary” sequences may also include, or be formed entirely from, non- Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
  • non- Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.
  • a polynucleotide that is "substantially complementary to at least part of a IncRNA transcript refers to a polynucleotide that is substantially complementary to a contiguous portion of the IncRNA of interest (e.g., an IncRNA transcript or gene).
  • a polynucleotide is complementary to at least a part of IncRNA if the sequence is substantially complementary to a non- interrupted portion of a IncRNA transcript or gene.
  • G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively.
  • ribonucleotide or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety.
  • guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil.
  • nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine.
  • a representation of an oligonucleotide as DNA may also be construed as RNA if the "T" nucleotides of the DNA are replaced in the sequence representation by "U".
  • adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target IncRNA.
  • US Patent 7,732,593 describes constructs forming G-Uwobble base pairs and is incorporated herein by reference in its entirety. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.
  • RNA molecule or "ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art.
  • a "ribonucleoside” includes a nucleoside base and a ribose sugar
  • a "ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties.
  • the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein.
  • the RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below.
  • an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2'-0-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, 5' phosphate group, 5' triphosate group, 5' phosphorodithioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino-modified nucleoside, 2'-alkyl-modified nucleoside, 2'- alkoxyalkyl-modified nucleoside e.g., (2'-0-methoxy ethyl) nucleoside
  • an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the molecule.
  • the modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule.
  • modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway or inhibit the function by steric effects such as translation arrest or modulation.
  • PNAs peptide nucleic acids
  • a modified ribonucleoside includes a deoxyribonucleoside.
  • an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA.
  • iRNA double stranded DNA molecule encompassed by the term "iRNA.”
  • an RNA interference agent includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA.
  • iRNA agent RNA interference agent
  • the invention relates to a single stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene, i.e., ssRNAi.
  • nucleotide overhang refers to at least one unpaired nucleotide that protrudes from the duplex structure of a LDA, e.g., a dsRNA, siRNA or generally an iRNA agent.
  • a dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more.
  • a nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • the overhang(s) may be on the sense strand, the antisense strand or any combination thereof.
  • the nucleotide(s) of an overhang can be present on the 5' end , 3' end or both ends of either an antisense or sense strand of a dsRNA.
  • the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3 ' end and/or the 5' end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3 ' end and/or the 5' end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
  • the sense strand of a dsRNA is connected with a biocleavable or biossable 1-25 nucleotide overhang at the 3 ' end and/or the 5' end capable of activating RNAse H.
  • one or more of the nucleotides in the overhang is replaced with a nucleotide sequence functioning as an immunostimuatory agent or as an aptamer.
  • the 5 '-end of the sense strand or antisense stand or both strands carry a triphosphate capable of activating RIG-I protein.
  • dsRNA dsRNA that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang.
  • One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended.
  • a "blunt ended" dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.
  • antisense strand or "guide strand” refers to the strand of an iR A agent, e.g., a dsRNA or siR A, which includes a region that is substantially complementary to a target sequence.
  • region of complementarity refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus. However, mismatches may be located in the internal positions of the molecule and on either strand of a dsRNA molecule.
  • sense strand refers to the strand of a iRNA agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
  • Double-stranded ribonucleic acid dsRNA
  • double-stranded RNA refers to an LDA that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having "sense” and “antisense” orientations with respect to a target RNA.
  • the duplex region can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 50 base pairs in length, e.g., 15-30 base pairs in length.
  • the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 46, 47, 48, 49 or 50 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs
  • dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length.
  • One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA.
  • the two strands forming the duplex structure can be from a single RNA molecule having at least one self- complementary region, or can be formed from two or more separate RNA molecules.
  • the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a "hairpin loop") between the 3 '-end of one strand and the 5 '-end of the respective other strand forming the duplex structure.
  • the hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
  • RNA molecules where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a "linker.”
  • linker the connecting structure
  • siRNA while being an iRNA agent may also used herein to refer to a dsRNA as described above.
  • the LDA agent includes double-stranded ribonucleic acid
  • dsRNA dsRNA molecules for inhibiting the expression of a IncRNA gene in a cell or mammal, e.g., in a human having a cancer or infectious disease
  • the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an RNA formed in the expression of a IncRNA, and where the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length
  • the dsRNA upon contact with a cell expressing the IncRNA, inhibits the expression of the IncRNA by at least 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method (to measure the proteins produced by any of the nearest neighbor genes or other protein coding gene known or believed to have a regulatory connection not a IncRNA), such as by Western blot.
  • the LDA agent activates the expression of a IncRNA in a cell or mammal.
  • Expression of a IncRNA in cell culture such as in COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells or in a biological sample from a subject can be assayed by measuring IncRNA RNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels of an associated protein coding gene (e.g., one indicative of IncRNA transcript levels), such as by immunofluorescence analysis, using, for example, Western Blotting or
  • a dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used.
  • One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an RNA formed during the expression of a IncRNA gene.
  • the other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.
  • the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive.
  • the region of complementarity to the target sequence. is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive.
  • the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive.
  • RNA targeted for cleavage will most often be part of a larger RNA molecule, often an IncRNA molecule.
  • a "part" of an IncRNA target is a contiguous sequence of an IncRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).
  • dsRNAs having duplexes as short as 9 base pairs can, under some
  • RNAi-directed RNA cleavage Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
  • the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of 9 to 36, e.g., 15-30 base pairs.
  • a dsRNA e.g., a duplex region of 9 to 36, e.g., 15-30 base pairs.
  • an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA.
  • a LDA agent useful to target IncRNA expression is generated in the target cell by cleavage of a larger dsRNA.
  • a dsRNA as described herein may further include one or more single-stranded nucleotide overhangs.
  • the dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are
  • a IncRNA is a human IncRNA.
  • the IncRNA is a mouse or a rat IncRNA.
  • the IncRNA is a cellular IncRNA to be targeted as a component or step in a bioprocessing reaction.
  • the first sequence is a sense strand of a dsRNA that includes a sense sequence referenced in Table 1, and the second sequence is selected from the group consisting of the corresponding antisense sequences of Table 1, the pairs of which are reported along with SEQ ID Nos of each sense:antisense pair. Pairs are listed with the sense strand first and then the antisense strand.
  • SEQ ID NO: 1 is the sense strand and SEQ ID NO: 2 is the antisense strand.
  • SEQ ID NO: 1 is the sense strand
  • SEQ ID NO: 2 is the antisense strand.
  • Each IncRNA transcript (prefix “ENST”) and IncRNA gene (prefix “ENSG”) from which the dsRNA are designed are also disclosed in Table 1.
  • a dsRNA will include one or more dsRNA nucleotide sequences, whereby the sense strand is selected from the groups of sequences provided in Table 1 the corresponding antisense strand of the sense strand selected from Table 1.
  • one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an RNA generated in the expression of a IncRNA.
  • a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Table 1, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand from Table 1.
  • the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.
  • RNA duplex structures having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et ah, EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective as well.
  • oligonucleotide sequences provided in Table 1 described herein can include at least one strand of a length of minimally 21 nt.
  • dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, or more contiguous nucleotides from one of the sequences of Table 1, and differing in their ability to inhibit the expression of a IncRNA by not more than 5, 10, 15, 20, 25, or 30 % inhibition from a dsRNA comprising the full sequence are contemplated according to the invention.
  • RNAs provided in Table 1 identify a site in a IncRNA transcript that is susceptible to RISC-mediated cleavage.
  • the present invention further features iRNAs that target within one of such sequences.
  • an iRNA agent is said to target within a particular site of an RNA transcript if the iRNA agent promotes cleavage of the transcript anywhere within that particular site.
  • Such an iRNA agent will generally include at least 15 contiguous nucleotides from one of the sequences provided in Table 1 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a IncRNA. Given the identification of the sites for targeting provided by Table 1, it is also within the scope of the present invention for an LDA to target substantially the same location or site.
  • target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA.
  • Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a "window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences.
  • the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected.
  • This process coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression.
  • sequences identified for example, in Table 1 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively "walking the window" one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
  • optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
  • modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
  • An iRNA agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA agent as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA agent contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA agent contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5' or 3 ' end of the region of complementarity.
  • the antisense strand generally does not contain any mismatch within the central 13 nucleotides.
  • the methods described herein or methods known in the art can be used to determine whether an iRNA agent containing a mismatch to a target sequence is effective in inhibiting the expression of a IncRNA. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a IncRNA is important, especially if the particular region of complementarity in a IncRNA is known to have polymorphic sequence variation within the population.
  • a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts.
  • the RNA of a LDA e.g., a dsRNA
  • the nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al.
  • Modifications include, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.) 3 ' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.) 3 ' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.)
  • base modifications e.g.
  • RNA compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages.
  • RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • the modified RNA will have a phosphorus atom in its internucleoside backbone.
  • Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and
  • thionoalkylphosphotriesters having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5, 166,315; 5, 185,444; 5,214, 134; 5,216, 141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;
  • RNA mimetics suitable or contemplated for use in iRNAs both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones and in particular— CH 2 — NH— CH 2 — , -CH 2 --N(CH 3 )--0--CH 2 - [known as a methylene (methylimino) or MMI backbone], ⁇ CH 2 ⁇ 0- -N(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -N(CH 3 )-CH 2 -CH 2 - [wherein the native phosphodiester backbone is represented as— O— P— O— CH 2 — ] of the above-referenced U.S.
  • RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
  • Modified RNAs may also contain one or more substituted sugar moieties.
  • the LDAs, e.g., dsRNAs, featured herein can include one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Exemplary suitable modifications include 0[(CH 2 ) n O] m CH 3 , 0(CH 2 ). n OCH 3 ,
  • n and m are from 1 to about 10.
  • dsRNAs include one of the following at the 2' position: Ci to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 ,
  • heterocycloalkyl heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the
  • the modification includes a 2'-methoxyethoxy (2'-0— CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et ah, Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
  • Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a 0(CH 2 ) 2 0N(CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0-CH 2 -0-CH 2 -N(CH 3 )2, also described in examples herein below.
  • 2'-dimethylaminooxyethoxy i.e., a 0(CH 2 ) 2 0N(CH 3 ) 2 group
  • 2'-DMAOE 2'-dimethylaminoethoxyethoxy
  • 2'-DMAEOE 2'-dimethylaminoethoxyethoxy
  • modifications include 2'-methoxy (2'-OCI3 ⁇ 4), 2'-aminopropoxy (2'- OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the RNA of a LDA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl, cyclohexenyl (CeNA), Hexose (HNA), FHNA moieties in place of the pentofuranosyl sugar. Representative U.S.
  • An iR A agent may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5 -hydroxy methyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5- trifluoromethyl
  • nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et ah, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993.
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention.
  • These include 5 -substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar
  • the RNA of a LDA can also be modified to include one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo structural conformation.
  • bicyclic nucleic acids, carbocyclic LNAs and amino LNAs are also within the present invention.
  • the addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J.
  • RNA of a LDA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the LDA.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem. Let., 1994, 4: 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al, Ann. N.Y.
  • Acids Res., 1990, 18:3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra ei a/., Biochim. Biophys. Acta, 1995, 1264:229- 237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923-937).
  • a ligand alters the distribution, targeting or lifetime of a LDA agent into which it is incorporated.
  • a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
  • Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
  • Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid.
  • the ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.
  • polyamino acids examples include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L- lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, PBAVE polymers with or without CDM linkers or polyphosphazine.
  • PLL polylysine
  • poly L-aspartic acid poly L-glutamic acid
  • styrene-maleic acid anhydride copolymer poly(L- lactide-co-glycolied) copolymer
  • polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide- polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
  • Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectins, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a cell or tissue targeting agent e.g., a lectins, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl- galactos amine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate,
  • polyaspartate a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.
  • These ligands can be either monovalent or polyvalent.
  • ligands include dyes, intercalating agents (e.g. acridines), cross- linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g.
  • intercalating agents e.g. acridines
  • cross- linkers e.g. psoralene, mitomycin C
  • porphyrins TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g.
  • EDTA lipophilic molecules, e.g, cholesterol, cholic acid, lithocholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino,
  • biotin e.g., aspirin, Ibuprofen, Naproxen, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
  • Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
  • Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactos amine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose.
  • the ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.
  • the ligand can be a substance, e.g, a drug, which can increase the uptake of the LDA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments.
  • the drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
  • the ligand is a lipid or lipid-based molecule.
  • a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA).
  • HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body.
  • the target tissue can be the liver, including parenchymal cells of the liver.
  • Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used.
  • a lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
  • a serum protein e.g., HSA.
  • a lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue.
  • a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body.
  • a lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
  • the lipid based ligand binds HSA.
  • it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non- kidney tissue.
  • the affinity it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
  • the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney.
  • Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
  • the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell.
  • a target cell e.g., a proliferating cell.
  • vitamins include vitamin A, E, and K.
  • Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.
  • the ligand is a cell-permeation agent, preferably a helical cell- permeation agent.
  • the agent is amphipathic.
  • An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids.
  • the helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
  • the ligand can be a peptide or peptidomimetic.
  • a peptidomimetic also referred to herein as an oligopeptidomimetic
  • a peptidomimetic is a molecule capable of folding into a defined three- dimensional structure similar to a natural peptide. The attachment of peptide and
  • peptidomimetics to IncRNA-directed agents can affect pharmacokinetic distribution of the LDA, such as by enhancing cellular recognition and absorption.
  • the peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • a peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe).
  • the peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide.
  • the peptide moiety can include a hydrophobic membrane translocation sequence (MTS).
  • An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et ah, Cancer Res., 62:5139-43, 2002).
  • An RGD peptide can facilitate targeting of an LDA to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et ah, Cancer Gene Therapy 8:783-787, 2001).
  • the RGD peptide will facilitate targeting of a LDA agent to the kidney.
  • the RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues.
  • a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing ayfi 3 (Haubner et ah, Jour. Nucl. Med., 42:326-336, 2001).
  • RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313;
  • the present invention also includes iRNA compounds that are chimeric compounds.
  • "Chimeric" iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound.
  • iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the LDA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the LDA may serve as a substrate for enzymes capable of cleaving
  • RNA:DNA or RNA:RNA hybrids RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the IncRNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • the RNA of a LDA can be modified by a non-ligand group.
  • a number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the LDA, and procedures for performing such conjugations are available in the scientific literature.
  • Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al, Biochem. Biophys. Res. Comm., 2007, 365(1):54-61 ; Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid
  • Acids Res., 1990, 18:3777 a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923).
  • RNA conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.
  • An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5 ' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
  • an iRNA agent will preferably have one or more of the following properties:
  • RNA-like properties i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content.
  • an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2' fluoro in place of 2' hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties.
  • the electronegative fluorine prefers an axial orientation when attached to the C2' position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a Cy-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix.
  • fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures.
  • a modified moiety at the 2' sugar position will be able to enter into H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide.
  • a preferred iRNA agent will: exhibit a Cy-endo pucker in all, or at least 50, 75,80, 85, 90, or 95 % of its sugars; exhibit a Cy-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, orl sugar which is not a Cy-endo pucker structure.
  • RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70 % of the nucleotides in the molecule, or more than 50, 60, or 70 % of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2' position, are excluded from the definition of RNA agent.
  • a "single strand iRNA agent” as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure.
  • Single strand iRNA agents are preferably antisense with regard to the target molecule.
  • single strand iRNA agents are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus.
  • 5'- phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5 '-monophosphate ((HO)2(0)P-0-5'); 5 '-diphosphate
  • iRNA agent is a single strand it is particularly preferred that it include a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
  • a single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target IncRNA.
  • a single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50 nnucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.
  • Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs.
  • the duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.
  • the hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3', and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length.
  • RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates.
  • Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body.
  • the art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et ah, (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196.
  • modified RNA refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified "RNAs,” they will of course, because of the modification, include molecules which are not RNAs.
  • Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of all of the above are discussed herein.
  • a double stranded iRNA agent e.g., a partially double stranded iRNA agent
  • double stranded structures e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)
  • intramolecular pairing e.g., a hairpin structure
  • nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety.
  • the modification will occur at all of the subject positions in the nucleic acid but in many, and infact in most cases it will not.
  • a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand.
  • a modification may occur in a double strand region, a single strand region, or in both.
  • a modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA.
  • a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini.
  • the 5' end or ends can be phosphorylated.
  • all or some of the bases in a 3 ' or 5' overhang will be modified, e.g., with a modification described herein.
  • Modifications can include, e.g., the use of modifications at the 2' OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence.
  • the scaffold presented above in Formula 1 represents a portion of a ribonucleic acid.
  • the basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers.
  • the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine
  • the sugars are the unmodified 2' hydroxyl ribose sugar (as depicted) and W, X, Y, and Z are all O
  • Formula 1 represents a naturally occurring unmodified
  • Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases.
  • Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Umodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent.
  • Modified nucleic acids and nucleotide surrogates can include one or more of:
  • modification of the 3' end or 5' end of the RNA e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g. a fluorescently labeled moiety, to either the 3' or 5' end of RNA.
  • the phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i.e., X and Y in Formula 1 above). However, the phosphate group can be modified by replacing one of the oxygens with a different substituent. One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
  • modified phosphate groups include phosphorothioate,
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which the individual diastereomers exhibit varying resistance to nucleases.
  • RNA containing chiral phosphate groups can be lower relative to the corresponding unmodified RNA species.
  • modifications to both X and Y which eliminate the chiral center, e.g. phosphorodithioate formation may be desirable in that they cannot produce diastereomer mixtures.
  • X can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).
  • Y can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred.
  • the phosphate linker can also be modified by replacement of a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged
  • a modified RNA can include modification of all or some of the sugar groups of the ribonucleic acid.
  • the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. While not being bound by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2' alkoxide ion.
  • the 2' alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.
  • MOE methoxyethyl group
  • OCH 2 CH 2 OCH 3 a PEG derivative
  • alkylamino dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino
  • -NHC(0)R alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar
  • R alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar
  • cyano alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar
  • cyano alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality.
  • Preferred substitutents are 2'- methoxyethyl, 2'-OCH3, 2'-0-allyl, 2'-C- allyl, and 2'-fluoro.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified RNA can include nucleotides containing e.g., arabinose, as the sugar.
  • Modified RNA's can also include "abasic" sugars, which lack a nucleobase at C- ⁇ . These abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms.
  • the 2' modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate).
  • phosphate linker modifications e.g., phosphorothioate
  • chimeric oligonucleotides are those that contain two or more different modifications.
  • the modificaton can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs.
  • the phosphate group can be replaced by non-phosphorus containing connectors (cf. Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.
  • moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
  • Preferred replacements include the methylenecarbonylamino and methylenemethylimino groups.
  • Oligonucleotide- mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone.
  • Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
  • a preferred surrogate is a PNA surrogate.
  • the 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end, 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group.
  • the 3' and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester).
  • labeling moieties e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester).
  • the functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer.
  • the terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3 ' or C-5' O, N, S or C group of the sugar.
  • the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs).
  • this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent.
  • the 3 ' end can be an - OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g.
  • psoralene mitomycin C
  • porphyrins TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g.
  • EDTA lipophilic carriers
  • lipophilic carriers e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino
  • biotin e.g., aspirin, vitamin E, folic acid
  • transport/absorption facilitators e.g., aspirin, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles.
  • Terminal modifications can be added for a number of reasons, including as discussed elsewhere herein to modulate activity or to modulate resistance to degradation.
  • Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or phosphate analogs.
  • iR A agents, especially antisense strands are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus.
  • 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
  • Suitable modifications include: 5 '-monophosphate ((HO)2(0)P-0-5'); 5 '-diphosphate ((HO)2(0)P-0- P(HO)(0)-0-5'); 5 '-triphosphate ((HO)2(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5'-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'- adenosine cap (Appp), and any modified or unmodified nucleotide cap structure ( -O-5'- (HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'-monothiophosphate (phosphorothioate;
  • HO2(S)P-0-5' 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-0-5'), 5'- phosphorothiolate ((HO)2(0)P-S-5'); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g.
  • Terminal modifications useful for increasing resistance to degradation include
  • Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross- linking an RNA agent to another moiety; modifications useful for this include mitomycin C.
  • Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties.
  • nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications.
  • substituted or modified analogs of any of the above bases e.g., "unusual bases" and "universal bases,” can be employed.
  • Examples include without limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines
  • base changes are less preferred for promoting stability, but they can be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent. Modified bases can reduce target specificity. This should be taken into consideration in the design of iRNA agents.
  • RNA agent e.g., a modified RNA
  • a candidate RNA agent for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property.
  • resistance to a degradent can be evaluated as follows.
  • a candidate modified RNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu, which includes a degradative agent, e.g., a nuclease.
  • a biological sample e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells.
  • the candidate and control could then be evaluated for resistance to degradation by any of a number of approaches.
  • the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or Cy5.
  • Control and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent.
  • a physical parameter, e.g., size, of the modified and control molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule.
  • a functional assay can also be used to evaluate the candidate agent.
  • a functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence lncRNA expression.
  • a cell e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914).
  • a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added.
  • Efficacy of the candidate agent on gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents.
  • oligoribonucleotides and oligoribonucleosides used in accordance with this invention may be with solid phase synthesis, see for example "Oligonucleotide synthesis, a practical approach", Ed. M. J. Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine- aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2'-0— Methyloligoribonucleotide- s: synthesis and applications, Chapter 4,
  • Phosphorothioate oligonucleotides Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2'-deoxyribonucleoside methylphosphonates, and. Chapter 7, Oligodeoxynucleotides containing modified bases.
  • Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223- 2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references referred to therein.
  • oligoribonucleotides is described in U.S. Pat. No. 5,023,243.
  • the preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5, 130,302 and 5, 177, 198.
  • the preparation of 3'-Deoxy-3'-amino phosphoramidate oligoribonucleotides is described in U.S. Pat. No. 5,476,925.
  • 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.
  • Preparation of sulfur bridged nucleotides is described in Sproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. Tetrahedron Lett. 1989, 30, 4693.
  • MMI linked oligoribonucleosides also identified herein as MMI linked oligoribonucleosides
  • methylenedimethylhydrazo linked oligoribonucleosides also identified herein as MDH linked oligoribonucleosides
  • methylenecarbonylamino linked oligonucleosides also identified herein as amide-3 linked oligoribonucleosides
  • methyleneaminocarbonyl linked oligonucleosides also identified herein as amide-4 linked oligoribonucleosides as well as mixed backbone compounds having, as for instance, alternating MMI and PO or PS linkages can be prepared as is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and in published PCT applications PCT/US92/04294 and
  • Cyclobutyl sugar surrogate compounds can be prepared as is described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared as is described in U.S. Pat. No. 5,519, 134. Morpholino sugar surrogates can be prepared as is described in U.S. Pat. Nos.
  • PNAs Peptide Nucleic Acids
  • PNA Peptide Nucleic Acids
  • Terminal modifications are described in Manoharan, M. et al. Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and references therein.
  • N-2 substitued purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,459,255.
  • 3-Deaza purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,457, 191.
  • 5,6-Substituted pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,614,617.
  • 5-Propynyl pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,484,908. Additional references can be disclosed in the above section on base modifications.
  • RNA agents have the following structure (see Formula 2 below):
  • R 1 , R 2 , and R 3 are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino
  • R 4 , R 5 , and R 6 are each, independently, OR 8 , 0(CH 2 CH 2 0) m CH 2 CH 2 OR 8 ; 0(CH 2 ) n R 9 ; 0(CH 2 ) n OR 9 , H; halo; NH 2 ; NHR 8 ; N(R 8 ) 2 ; NH(CH 2 CH 2 NH) m CH 2 CH 2 NHR 9 ; NHC(0)R 8 ; ; cyano; mercapto, SR 8 ; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialky
  • alkanesulfonamido arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or R 4 , R 5 , or R 6 together combine with R 7 to form an [-0-CH 2 -] covalently bound bridge between the sugar 2' and 4' carbons.
  • a 1 is:
  • a preferred Al is chosen from 5'- monophosphate (( ⁇ ) 2 (0) ⁇ -0-5'), 5'-diphosphate (( ⁇ ) 2 (0) ⁇ -0- ⁇ ( ⁇ )(0)-0-5'), 5'- triphosphate (( ⁇ ) 2 (0) ⁇ -0-( ⁇ )(0) ⁇ -0- ⁇ ( ⁇ )(0)-0-5'), 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5'-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'), 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-0-5'-(HO)(0)P-0- ( ⁇ )(0) ⁇ -0- ⁇ ( ⁇ )(0)-0-5'), 5'-monothiophosphate (phosphorothioate; (HO) 2 (S)P-0-5'), 5'- monodithiophosphate (phosphorodithio
  • any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.
  • 5'-phosphoramidates (( ⁇ ) 2 (0) ⁇ - ⁇ -5', ( ⁇ )( ⁇ 2 )(0) ⁇ -0-5'), 5'-alkylphosphonates
  • a 2 is:
  • A is:
  • a 4 is:
  • W 1 is OH, (CH 2 ) n R 10 , (CH 2 ) n NHR 10 , (CH 2 ) n OR 10 , (CH 2 ) n SR 10 ; 0(CH 2 ) n R 10 ;
  • X 1 , X 2 , X 3 , and X 4 are each, independently, O or S.
  • Y 1 , Y 2 , Y 3 , and Y 4 are each, independently, OH, O “ , OR 8 , S, Se, BH 3 " , H, NHR 9 , N(R 9 ) 2 alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.
  • Z 1 , Z 2 , and Z 3 are each independently O, CH 2 , NH, or S.
  • Z 4 is OH, (CH 2 ) n R 10 , (CH 2 ) n NHR 10 , (CH 2 ) n OR 10 , (CH 2 ) n SR 10 ; 0(CH 2 ) n R 10 ; 0(CH 2 ) n OR 10 , 0(CH 2 ) n NR 10 ,
  • x is 5-100, chosen to comply with a length for an RNA agent described herein.
  • R 7 is H; or is together combined with R 4 , R 5 , or R 6 to form an [-0-CH 2 -] covalently bound bridge between the sugar 2' and 4' carbons.
  • R 8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar;
  • R 9 is NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and
  • R is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), cross-linkers (e.g.
  • psoralene mitomycin C
  • porphyrins TPPC4,texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g. EDTA
  • lipohilic carriers cholesterol, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group,
  • peptide conjugates e.g., antennapedia peptide, Tat peptide
  • alkylating agents phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes, haptens (e.g.
  • biotin e.g., aspirin, vitamin E, folic acid
  • transport/absorption facilitators e.g., aspirin, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of
  • RNA agent m is 0-1,000,000, and n is 0-20.
  • Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents.
  • Preferred iRNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):
  • a 10 -A 40 is L-G-L; A 10 and/or A 40 may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH 2 (CH 2 ) g ; N(CH 2 ) g ; 0(CH 2 ) g ; S(CH 2 ) g .
  • G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and
  • R 10 , R 20 , and R 30 are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5- halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,
  • R 40 , R 50 , and R 60 are each, independently, OR 8 , 0(CH 2 CH 2 0) m CH 2 CH 2 OR 8 ;
  • NHC(0)R 8 ;; cyano; mercapto, SR 7 ; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsul
  • x is 5-100 or chosen to comply with a length for an RNA agent described herein.
  • RTM is H; or is together combined with R 40 , R 50 , or R 60 to form an [-0-CH 2 -] covalently bound bridge between the sugar 2' and 4' carbons.
  • R 8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and R 9 is NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid, m is 0-1,000,000, n is 0-20, and g is 0-2.
  • Preferred nucleoside surrogates have the following structure (see Formula 4 below):
  • S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid.
  • L is a linker and is selected from the group consisting of CH 2 (CH 2 ) g ; N(CH 2 ) g ; 0(CH 2 ) g ; S(CH 2 ) g ; -C(0)(CH 2 ) n -or may be absent.
  • M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent.
  • R , R , and R are each, independently, H (i.e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5- halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thio
  • x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2.
  • the invention features a nuclease resistant monomer, or a an iRNA agent which incorporates a nuclease resistant monomer (NMR), such as those described herein and those described in copending, co-owned United States Provisional Application Serial No.
  • NMR nuclease resistant monomer
  • the invention includes iRNA agents having a NMR and another element described herein.
  • the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a NMR.
  • an iRNA agent described herein e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing
  • an iRNA agent which targets a gene described herein e.g., a gene active in the liver
  • an iRNA agent having an architecture or structure described herein
  • An iRNA agent can include monomers which have been modifed so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRM's, or nuclease resistance promoting monomers or modifications.
  • RNA-induced Silencing Complex RNA-induced Silencing Complex
  • modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex.
  • Preferred modifications are those that increase exonuclease and endonuclease resistance and thus prolong the halflife of the iRNA agent prior to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex.
  • An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the modifications described herein.
  • the anti sense strand may include modifications at the 3' end and/or the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand.
  • the sense strand may include modifications at the 3 ' end and/or the 5' end and/or at any one of the intervening positions between the two ends of the strand.
  • the iRNA agent may also include a duplex comprising two hybridized antisense strands.
  • the first and/or the second antisense strand may include one or more of the modifications described herein.
  • one and/or both antisense strands may include modifications at the 3' end and/or the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular configurations are discussed below.
  • Modifications that can be useful for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone:
  • preferred NRM's include nucleotide dimers with an enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., Sp or Rp, at the position X, where this is the position normally occupied by the oxygen.
  • the atom at X can also be S, Se, Nr 2 , or Br 3 .
  • X is S
  • enriched or chirally pure Sp linkage is preferred.
  • Enriched means at least 70, 80, 90, 95, or 99% of the preferred form.
  • preferred NRM's include monomers at the terminal position derivitized at a cationic group.
  • this NRM is preferraly not used at th 5' end of an anti-sense sequence.
  • the group should be attached at a position on the base which minimizes intererence with H bond formation and hybridization, e.g., away form the face which intereacts with the complementary base on the other strand, e.g, at the 5' position of a pyrimidine or a 7-position of a purine.
  • L-RNA, 2'-5' likages, inverted linkages, a-nucleosides are preferred NRM's.
  • other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2'-5' phosphate, non-phosphate and modified phosphate linkages (e.g., thiophospahtes,
  • dimers having inverted linkages e.g., 3 '-3' or 5'-5' linkages
  • monomers having an alpha linkage at the site on the sugar e.g., the structures described herein having an alpha linkage
  • preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar , base, or backbone ;
  • preferred NRM's can include an abasic monomer, e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein.; and
  • preferred NRM's include monomers, preferably at the terminal position, e.g., the 5' position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result of the action of a component in the subject's body, e.g, a carboxyesterase or an enzyme present in the subject's body.
  • a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which attacks a carbon adjacent to the O of a phosphate and resulting in the production of an uprotected phosphate.
  • NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent.
  • An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintainted.
  • NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off-target silencing.
  • a modification can include the alteration, e.g., replacement, of one or both of the non- linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens.
  • Formula X depicts a phosphate moiety linking two sugar/sugar surrogate-base moities, SBi and SB 2 .
  • one of the non-linking phosphate oxygens in the phosphate backbone moiety can be replaced by any one of the following: S, Se, BR 3 (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR 2 (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl).
  • S, Se R is hydrogen, alkyl, aryl, etc.
  • C i.e., an alkyl group, an aryl group, etc.
  • H NR 2
  • OR R is alkyl or aryl
  • the phosphorus atom in an unmodified phosphate group is achiral.
  • the stereogenic phosphorus atom can possess either the "R" configuration (herein Rp) or the "S” configuration (herein Sp).
  • Rp the "R" configuration
  • Sp the "S” configuration
  • iRNA agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the Sp configuration.
  • these atoms e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms
  • iRNA agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the Rp configuration.
  • the population of stereogenic phosphorus atoms may have the Sp configuration and may be substantially free of stereogenic phosphorus atoms having the Rp configuration.
  • the population of stereogenic phosphorus atoms may have the Rp configuration and may be substantially free of stereogenic phosphorus atoms having the Sp configuration.
  • substantially free of stereogenic phosphorus atoms having the Rp configuration means that moieties containing stereogenic phosphorus atoms having the Rp configuration cannot be detected by conventional methods known in the art (chiral HPLC, l H NMR analysis using chiral shift reagents, etc.).
  • the phrase "substantially free of stereogenic phosphorus atoms having the Sp configuration” means that moieties containing stereogenic phosphorus atoms having the Sp configuration cannot be detected by conventional methods known in the art (chiral HPLC, X H NMR analysis using chiral shift reagents, etc.).
  • modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom.
  • the population of phosphorothioate stereogenic phosphorus atoms may have the Sp configuration and be substantially free of stereogenic phosphorus atoms having the R P configuration.
  • Phosphorothioates may be incorporated into iR A agents using dimers e.g., formulas X-l and X-2. The former can be used to introduce phosphorothioate
  • Y can be 2-cyanoethoxy
  • W and Z can be O
  • !3 ⁇ 4' can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH 3 )
  • DMT is dimethoxytrityl
  • "BASE" can be a natural, unusual, or a universal base.
  • X-l and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms having essentially only the Rp configuration (i.e., being substantially free of the Sp configuration) or only the Sp configuration (i.e., being substantially free of the Rp configuration).
  • dimers can be prepared having a population of stereogenic phosphorus atoms in which about 50% of the atoms have the Rp configuration and about 50% of the atoms have the Sp
  • Dimers having stereogenic phosphorus atoms with the Rp configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the Sp configuration using e.g., enzymatic degradation and/or conventional chromatography techniques.
  • Modifications can also include attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety.
  • a cationic group can be attached to any atom capable of substitution on a natural, unusual or universal base.
  • a preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing.
  • a cationic group can be attached e.g., through the C2' position of a sugar or analogous position in a cyclic or acyclic sugar surrogate.
  • Modifications can also include the incorporation of nonphosphate linkages at the 5' and/or 3' end of a strand.
  • nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo,
  • methylenedimethylhydrazo and methyleneoxymethylimino Preferred replacements include the methyl phosphonate and hydroxylamino groups.
  • modifications can include replacement of one of the bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom..
  • Modifications can also include linking two sugars via a phosphate or modified phosphate group through the 2' position of a first sugar and the 5' position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are eached linked through the respective3' positions.
  • Modified RNA's can also include "abasic" sugars, which lack a nucleobase at C- .
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar.
  • the natural, unusual, or universal base may have the a-configuration.
  • Modifcations can also include L-RNA.
  • the prodrug groups may be decomposed via reaction first with carboxy esterases. The remaining ethyl thiolate group via intramolecular SN2 displacement can depart as episulfide to afford the underivatized phosphate group.
  • Modification can also include the addition of conjugating groups described elseqhere herein, which are prefereably attached to an iRNA agent through any amino group available for conjugation.
  • Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit hybridization so it is preferably to use them only in terminal regions, and preferrable to not use them at the cleavage site or in the cleavage region of an sequence which targets a subject sequence or gene.. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some
  • embodiments it is desirabable to put the NRM at the cleavage site or in the cleavage region of a sequence which does not target a subject sequence or gene, as it can minimize off-target silencing.
  • an iRNA agent described herein can have an overhang which does not form a duplex structure with the other sequence of the iRNA agent— it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence of the iRNA agent.
  • nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which interfer with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et ah, 2001, Genes and Dev.
  • cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 1 1 nucleotides upstream of the first nucleotide which is complementary to the guide sequence.
  • cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it.
  • Cleavage region means an nucleotide with 1, 2, or 3 nucletides of the cleave site, in either direction.
  • Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject.
  • An iRNA agent can have a first and a second strand chosen from the following:
  • a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;
  • a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
  • a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
  • a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3 ' and the 5' end;
  • a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;
  • a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);
  • a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
  • a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;
  • a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3 ' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand).
  • An iRNA agent can also target two sequences and can have a first and second strand chosen from:
  • a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;
  • a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);
  • a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
  • a first strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;
  • a first strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand) and a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;
  • a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);
  • a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
  • a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;
  • a second strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3 ' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand).
  • the invention features a ribose mimic, or an iRNA agent which incorporates a ribose mimic, such as those described herein and those described in copending co- owned United States Provisional Application Serial No. 60/454,962 (Attorney Docket No.
  • the invention includes iRNA agents having a ribose mimic and another element described herein.
  • the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a ribose mimic.
  • an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent.
  • Nucleases e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid.
  • the secondary hydroxy group confers nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the modification.
  • a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3 ' ribose hydroxyl group, thereby causing it to be less susceptible to degradation.
  • the secondary hydroxyl group refers to an "OH" radical that is attached to a carbon atom substituted by two other carbons and a hydrogen.
  • the secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group.
  • the hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base..
  • the hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group.
  • Preferred iRNA agent delivery modules with five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group have the following structure (see formula Y below):
  • A is an iRNA agent, including any iRNA agent described herein.
  • the iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to "W" of the phosphate group.
  • the iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, and Z are O) or modified.
  • W and Z can be independently NH, O, or S; and
  • X and Y can be independently S, Se, BH 3 " , C ⁇ -Ce alkyl, C6-C1 0 aryl, H, O, O " , alkoxy or amino (including alkylamino, arylamino, etc.).
  • W, X and Z are O and Y is
  • Ri and R3 are each, independently, hydrogen; or C1-C1 00 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.
  • R2 is hydrogen; C1-C1 00 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R 2 may be taken together with with R4 or R ⁇ to form a ring of 5-12 atoms.
  • R4 is hydrogen; C1-C100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R4 may be taken together with with R2 or R5 to form a ring of 5-12 atoms.
  • R5 is hydrogen, C1-C1 00 alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R5 may be taken together with with R4 to form a ring of 5-12 atoms.
  • R6 is hydrogen, C1-C1 00 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when n is 1, R6 may be taken together with with R2 to form a ring of 6-10 atoms;
  • R 7 is hydrogen, C1-C1 00 alkyl, or C(0)(CH 2 ) q C(0)NHR 9 ;
  • T is hydrogen or a functional group;
  • n and q are each independently 1-100;
  • Rs is C1-C1 0 alkyl or C6-C1 0 aryl; and
  • R9 is hydrogen, CI -CIO alkyl, C6-C10 aryl or a solid support agent.
  • Preferred embodiments may include one of more of the following subsets of iRNA agent delivery modules.
  • A can be connected directly or indirectly through a terminal 3 ' or 5 ' ribose sugar carbon of the RNA agent.
  • RNAi agent delivery modules In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S.
  • n is 1, and R2 and R6 are taken together to form a ring containing six atoms and R4 and R5 are taken together to form a ring containing six atoms.
  • the ring system is a trans-dscal .
  • the RNAi agent delivery module of this subset can include a compound of Formula (Y-l):
  • the functional group can be, for example, a targeting group (e.g., a steroid or a carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety).
  • a targeting group e.g., a steroid or a carbohydrate
  • a reporter group e.g., a fluorophore
  • a label an isotopically labelled moiety
  • the targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin.
  • endothelial cell targeting groups e.g., RGD peptides and mimetics
  • cancer cell targeting groups e.g., folate Vitamin B12, Biotin
  • bone cell targeting groups e.g., bisphosphonates, polyglutamates, polyaspartates
  • multivalent mannose for e.g., macrophage testing
  • lactose galactose
  • Synthetic chemistry transformations and protecting group methodologies useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
  • iRNA agents can be modified in a number of ways which can optimize one or more characteristics of the iRNA agent.
  • the invention features a ribose replacement monomer subunit (RRMS), or a an iRNA agent which incorporates a RRMS, such as those described herein and those described in one or more of United States Provisional Application Serial No. 60/493,986 (Attorney Docket No. 14174-079P01), filed on August 8, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/494,597 (Attorney Docket No. 14174-080P01), filed on August 11, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No.
  • the invention includes iRNA agents having a RRMS and another element described herein.
  • the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an archtecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a RRMS.
  • an iRNA agent described herein e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing
  • an iRNA agent which targets a gene described herein e.g., a gene active in the liver
  • ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier.
  • a non-carbohydrate preferably cyclic
  • a cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur.
  • the cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings.
  • the cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.
  • the carriers further include (i) at least two "backbone attachment points” and (ii) at least one "tethering attachment point.”
  • a "backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid.
  • a "tethering attachment point" as used herein refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety.
  • the moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property, e.g., lipophilicity, of an iRNA agent.
  • the selected moiety is connected by an intervening tether to the cyclic carrier.
  • it will include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.
  • incorporación of one or more RRMSs described herein into an RNA agent can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance.
  • Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target IncRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).
  • the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (R- 1) is incorporated into at least one of said strands.
  • X is N(CO)R 7 , NR 7 or CH 2 ; Y is NR 8 , O, S, CR 9 R 10 , or absent; and Z is CR U R 12 or absent.
  • Each of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , OR b , (CH 2 ) n OR a , or (CH 2 ) n OR b , provided that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is OR a or OR b and that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is (CH 2 ) n OR a , or (CH 2 ) n OR b (when the RRMS is terminal, one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 will include R a and one will include R b ; when the RRMS is internal, two of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 will each include an R b ); further provided that preferably
  • Each of R 5 , R 6 , R 11 , and R 12 is, independently, H, Ci-C 6 alkyl optionally substituted with 1-3 R 13 , or C(0)NHR 7 ; or R 5 and R 11 together are C 3 -C 8 cycloalkyl optionally substituted with R 14 .
  • R 7 is Ci-C 20 alkyl substituted with NR c R d ;
  • R 8 is Ci-C 6 alkyl;
  • R 13 is hydroxy, d-C 4 alkoxy, or halo;
  • R 14 is NR C R 7 .
  • R a is:
  • R b is:
  • Each of A and C is, independently, O or S.
  • B is OH, O " , or
  • R c is H or C1-C6 alkyl
  • R d is H or a ligand
  • n is 1-4.
  • the ribose is replaced with a pyrroline scaffold, and X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent.
  • the ribose is replaced with a piperidine scaffold, and X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is CR n R 12 .
  • the ribose is replaced with a piperazine scaffold, and X is N(CO)R 7 or NR 7 , Y is NR 8 , and Z is CR n R 12 .
  • the ribose is replaced with a morpholino scaffold, and X is N(CO)R 7 or NR 7 , Y is O, and Z is CR n R 12 .
  • the ribose is replaced with a decalin scaffold, and X isCH 2 ; Y is CR 9 R 10 ; and Z is CR U R 12 ; and R 5 and R 11 together are C 6 cycloalkyl.
  • the ribose is replaced with a decalin/indane scafold and , and X is CH 2 ; Y is CR 9 R 10 ; and Z is CR n R 12 ; and R 5 and R 11 together are C 5 cycloalkyl.
  • the ribose is replaced with a hydroxyproline scaffold.
  • RRMSs described herein may be incorporated into any double-stranded RNA-like molecule described herein, e.g., an iRNA agent.
  • An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the RRMSs described herein.
  • An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent.
  • An RRMS can be placed at or near (within 1, 2, or 3 positions) of the 3 ' or 5' end of the sense strand or at near (within 2 or 3 positions of) the 3' end of the antisense strand.
  • an RRMS at or near (within 1, 2, or 3 positions of) the 5' end of the antisense strand.
  • An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.
  • an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3 ' end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and at (or within 1, 2, or 3 positions of) the 3 ' end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both ligands are located at the same end of the iRNA agent.
  • two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.
  • an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3 ' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand.
  • ligand e.g., cholic acid
  • a ligand shared between two proximal RRMSs is referred to herein as a "hairpin ligand.”
  • an iRNA agent may have an RRMS at the 3 ' end of the sense strand and an RRMS at an internal position of the sense strand.
  • An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.
  • the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme.
  • the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule.
  • a ds iRNA agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides. Most embodiments will have a 3 ' overhang.
  • Preferred sRNA agents will have single-stranded overhangs, preferably 3' overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5' ends are preferably phosphorylated.
  • Preferred carriers have the general formula (R-3) provided below.
  • R-3 preferred backbone attachment points can be chosen from R 1 or R 2 ; R 3 or R 4 ; or R 9 and R 10 if Y is CR 9 R 10 (two positions are chosen to give two backbone attachment points, e.g., R 1 and R 4 , or R 4 and R 9 .
  • Preferred tethering attachment points include R 7 ; R 5 or R 6 when X is CH 2 .
  • the carriers are described below as an entity, which can be incorporated into a strand.
  • the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R 1 or R 2 ; R 3 or R 4 ; or R 9 or R 10 (when Y is CR 9 R 10 ), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone.
  • one of the above-named R groups can be - CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.
  • X is N(CO)R 7 , NR 7 or CH 2 ; Y is NR 8 , O, S, CR 9 R 10 ; and Z is CR n R 12 or absent.
  • Each of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , or (CH 2 ) n OR b , provided that at least two of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 are OR a and/or (CH 2 ) n OR b .
  • Each of R 5 , R 6 , R 11 , and R 12 is, independently, a ligand, H, Ci-C 6 alkyl optionally substituted with 1-3 R 13 , or C(0)NHR 7 ; or R 5 and R 11 together are C 3 -C 8 cycloalkyl optionally substituted with R 14 .
  • R 7 is H, a ligand, or Ci-C 20 alkyl substituted with NR c R d ;
  • R 8 is H or Ci-C 6 alkyl;
  • R 13 is hydroxy, C 1 -C 4 alkoxy, or halo;
  • R 14 is NR C R 7 ;
  • R 15 is Ci-Ce alkyl optionally substituted with cyano, or C2-C6 alkenyl;
  • R 16 is C1-C1 0 alkyl; and
  • R 17 is a liquid or solid phase support reagent.
  • L is -C(0)(CH 2 ) q C(0)-, or -C(0)(CH 2 ) q S-;
  • R a is CAr 3 ;
  • R b is P(0)(0 " )H,
  • R c is H or Ci-C 6 alkyl
  • R d is H or a ligand.
  • Each Ar is, independently, C6-C1 0 aryl optionally substituted with C1-C4 alkoxy; n is 1-4; and q is 0-4.
  • the carrier may be based on the pyrroline ring system or the 3-hydroxyproline ring system, e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent (D).
  • OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene
  • D group e.g., a methylene group, connected to one of the carbons in the five-membered ring (- CH 2 OFG 1 in D).
  • OFG 2 is preferably attached directly to one of the carbons in the five- membered ring (-OFG 2 in D).
  • -CH 2 OFG 1 may be attached to C- 2 and OFG 2 may be attached to C-3; or -CH 2 OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4.
  • CH 2 OFG 1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.
  • -CH 2 OFG 1 may be attached to C-2 and OFG 2 may be attached to C-4.
  • the pyrroline- and 3-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • CHzOFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included.
  • the tethering attachment point is preferably nitrogen.
  • the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is CR U R 12 .
  • E piperidine ring system
  • X is N(CO)R 7 or NR 7
  • Y is CR 9 R 10
  • Z is CR U R 12 .
  • OFG 1 is preferably
  • OFG 2 is preferably attached directly to one of the carbons in the six-membered ring (-OFG 2 in E).
  • -(CH ⁇ n OFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4.
  • - (CH 2 ) n OFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH ⁇ n OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; -(CH ⁇ n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; -(CH 2 )nOFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or -(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-3.
  • the piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • -(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included.
  • the tethering attachment point is preferably nitrogen.
  • the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R 7 or NR 7 , Y is NR 8 , and Z is CR U R 12 , or the morpholine ring system (G), e.g., X is N(CO)R 7 or NR 7 , Y is O, and Z is CR n R 12 .
  • F piperazine ring system
  • G e.g., X is N(CO)R 7 or NR 7
  • Y is O
  • Z is CR n R 12 .
  • OFG 1 is preferably
  • a primary carbon e.g., an exocyclic alkylene group, e.g., a methylene group
  • OFG 2 is preferably attached directly to one of the carbons in the six-membered rings (-OFG 2 in F or G).
  • -CH 2 OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; or vice versa.
  • CH 2 OFG 1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.
  • the piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • CH 2 OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included.
  • R" ' can be, e.g., C ⁇ -Ce alkyl, preferably CH 3 .
  • the tethering attachment point is preferably nitrogen in both F and G.
  • OFG 1 is preferably attached to a primary carbon
  • OFG 2 is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG 2 in H).
  • -(CH ⁇ n OFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5.
  • -(CH ⁇ n OFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH 2 ) n OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3 ; -(CH ⁇ n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; -(CH ⁇ n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or - (CH ⁇ n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-3; -(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-5; or -(CH ⁇ n OFG 1 may be attached to C-5 and OFG 2 may be attached to C-4.
  • the decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • -(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included.
  • the substituents at C-l and C-6 are trans with respect to one another.
  • the tethering attachment point is preferably C-6 or C-l .
  • Other carriers may include those based on 3-hydroxyproline (J).
  • -(CH2) n OFG 1 and OFG 2 may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers
  • the tethering attachment point is preferably nitrogen.
  • a moiety e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether.
  • Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any Ci-Cioo carbon-containing moiety, (e.g. C1-C75, C1-C50, C1-C20, C1-C10, C1-C6), preferably having at least one nitrogen atom.
  • the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand.
  • Preferred tethers include TAP Z
  • n 1-6 and R" " is d- e alkyl. and R d is hydrogen or a ligand.
  • the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or hydrazino group, -NHNH2.
  • the tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S.
  • Preferred tethered ligands may include, e.g., TAP-(CH?) n NH(LIGAND) .
  • the tether may include an electrophilic moiety, preferably at the terminal position of the tether.
  • electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester.
  • Preferred tethers (underlined) include TAP Z
  • TAP-fCH CfOOC ⁇ Fs TAP-CtOYCH?) ⁇ ( ⁇ ) OCF or TAP-NR" "( ⁇ ⁇ OC fi F.. in which n is 1-6 and R"" is Ci-C 6 alkyl; or -fCHACH 7 LG: TAP-CfOYCH CIfrLG: or TAP- NR""(CHACH 7 LG. in which n is 1-6 and R"" is Ci-C 6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate).
  • Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.
  • a wide variety of entities can be tethered to an iRNA agent, e.g., to the carrier of an RRMS. Examples are described below in the context of an RRMS but that is only preferred, entities can be coupled at other points to an iRNA agent.
  • Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier.
  • the ligand is attached to the carrier via an intervening tether.
  • the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incorporated into the growing strand.
  • the ligand may be incorporated into a "precursor" RRMS after a "precursor" RRMS monomer has been incorporated into the growing strand.
  • an RRMS monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH 2 ) n NH 2 may be incorporated into a growing sense or antisense strand.
  • a ligand having an electrophilic group e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor RRMS by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor RRMS tether.
  • a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated.
  • a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
  • Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
  • Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.
  • Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties.
  • General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.
  • Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid.
  • the ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.
  • polyamino acids examples include polyamino acid is a poly lysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L- lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine.
  • PLL poly lysine
  • poly L-aspartic acid poly L-glutamic acid
  • styrene-maleic acid anhydride copolymer poly(L- lactide-co-glycolied) copolymer
  • divinyl ether-maleic anhydride copolymer divinyl
  • polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
  • Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell.
  • a cell or tissue targeting agent e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell.
  • a targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic.
  • ligands include dyes, intercalating agents (e.g. acridines), cross- linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g.
  • intercalating agents e.g. acridines
  • cross- linkers e.g. psoralene, mitomycin C
  • porphyrins TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g.
  • EDTA lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl,
  • biotin e.g., aspirin, vitamin E, folic acid
  • transport/absorption facilitators e.g., aspirin, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
  • Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
  • Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactos amine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose.
  • the ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.
  • the ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments.
  • the drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
  • the ligand can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example.
  • exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin- 1 beta, or gamma interferon.
  • the ligand is a lipid or lipid-based molecule.
  • a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA).
  • HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body.
  • the target tissue is the liver, preferably parenchymal cells of the liver.
  • Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used.
  • a lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a seru protein, e.g., HSA.
  • a lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue.
  • a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body.
  • a lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
  • the lipid based ligand binds HSA.
  • it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non- kidney tissue.
  • the affinity it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
  • the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney.
  • Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
  • the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell.
  • a target cell e.g., a proliferating cell.
  • vitamins include vitamin A, E, and K.
  • Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.
  • the ligand is a cell-permeation agent, preferably a helical cell- permeation agent.
  • the agent is amphipathic.
  • An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids.
  • the helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
  • the ligand can be a peptide or peptidomimetic.
  • a peptidomimetic also referred to herein as an oligopeptidomimetic is a molecule capable of folding into a defined three- dimensional structure similar to a natural peptide. The attachment of peptide and
  • peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption.
  • the peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • a peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe).
  • the peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide.
  • the peptide moiety can include a hydrophobic membrane translocation sequence (MTS).
  • An exemplary hydrophobic MTS-containing peptide is RFGF.
  • An RFGF analogue containing a hydrophobic MTS can also be a targeting moiety.
  • the peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes.
  • delivery peptide can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes.
  • sequences from the HIV Tat protein and the Drosophila Antennapedia protein have been found to be capable of functioning as delivery peptides.
  • a peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et ah, Nature, 354:82-84, 1991).
  • OBOC one-bead-one-compound
  • the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic.
  • RGD arginine-glycine-aspartic acid
  • a peptide moiety can range in length from about 5 amino acids to about 50 amino acids.
  • the peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
  • An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et ah, Cancer Res., 62:5139-43, 2002).
  • An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et ah, Cancer Gene Therapy 8:783-787, 2001).
  • the RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues.
  • a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing ayfi 3 (Haubner et ah, Jour. Nucl. Med., 42:326-336, 2001).
  • Peptides that target markers enriched in proliferating cells can be used.
  • RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an I v 3 ⁇ 4 integrin.
  • RGD one can use other moieties that target the I v -3 ⁇ 4 integrin ligand.
  • such ligands can be used to control proliferating cells and angiogeneis.
  • Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.
  • a "cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell.
  • a microbial cell- permeating peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., a -defensin, ⁇ -defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin).
  • a cell permeation peptide can also include a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV- 1 gp41 and the NLS of SV40 large T antigen (Simeoni et ah, Nucl. Acids Res. 31 :2717-2724, 2003).
  • a targeting peptide tethered to an RRMS can be an amphipathic a- helical peptide.
  • exemplary amphipathic a-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S.
  • clava peptides hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H 2 A peptides, Xenopus peptides, esculentinis- 1, and caerins.
  • HFIAPs hagfish intestinal antimicrobial peptides
  • magainines brevinins-2, dermaseptins, melittins, pleurocidin
  • H 2 A peptides Xenopus peptides, esculentinis- 1, and caerins.
  • H 2 A peptides Xenopus peptides
  • esculentinis- 1, and caerins esculentinis- 1, and caerins.
  • a number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala,
  • the capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H- bond to stabilize the helix.
  • Formation of salt bridges between residues with opposite charges, separated by i ⁇ 3, or i ⁇ 4 positions can provide stability.
  • cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.
  • Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; ⁇ , ⁇ , or ⁇ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
  • D or L peptides e.g., D or L peptides
  • ⁇ , ⁇ , or ⁇ peptides N-methyl peptides
  • azapeptides peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages
  • cyclic peptides e.g., D
  • iRNA agents can include modified or non-naturally occuring bases, e.g., bases described in copending and coowned United States Provisional Application Serial No.
  • the invention includes iRNA agents having a modified or non-naturally occuring base and another element described herein.
  • the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a modified or non-naturally occuring base.
  • oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan, "Oligonucleotide Conjugates in Antisense Technology," in Antisense Drug
  • a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide.
  • the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989).
  • An azapeptide is synthesized by replacing the a-carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain.
  • the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a "carbonyl donor," e.g., phenylchloroformate.
  • a peptide or peptidomimetic e.g., a peptide or peptidomimetic tethered to an RRMS
  • N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage.
  • N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21 :99, 2000; Cell Penetrating Peptides: Processes and
  • an Ant or Tat peptide can be an N-methyl peptide.
  • a peptide or peptidomimetic e.g., a peptide or peptidomimetic tethered to an RRMS
  • a peptide or peptidomimetic can be a ⁇ -peptide.
  • ⁇ -peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state, ⁇ -peptides are resistant to degradation by proteolytic enzymes, ⁇ -peptides can be synthesized by methods known in the art.
  • an Ant or Tat peptide can be a ⁇ -peptide.
  • a peptide or peptidomimetic e.g., a peptide or peptidomimetic tethered to an RRMS
  • a peptide or peptidomimetic can be a oligocarbamate.
  • Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters.
  • an Ant or Tat peptide can be an oligocarbamate.
  • a peptide or peptidomimetic e.g., a peptide or peptidomimetic tethered to an RRMS
  • a peptide or peptidomimetic can be an oligourea conjugate (or an oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety.
  • an oligourea conjugate is tethered to an iRNA agent for use in oral delivery.
  • the backbone in each repeating unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid.
  • the single carbon atom extension can increase peptide stability and lipophilicity, for example.
  • An oligourea peptide can therefore be advantageous when an iRNA agent is directed for passage through a bacterial cell wall, or when an iRNA agent must traverse the blood-brain barrier, such as for the treatment of a neurological disorder.
  • a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor.
  • an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate).
  • siRNA peptide conjugates of the invention can be affiliated with, e.g., tethered to, RRMSs occurring at various positions on an iRNA agent.
  • a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand).
  • the peptide can be internally conjugated, such as in the loop of a short hairpin iRNA agent.
  • the peptide can be affiliated with a complex, such as a peptide-carrier complex.
  • a peptide-carrier complex consists of at least a carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type.
  • a carrier complex can carry additional targeting molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery.
  • the one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier.
  • a carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer.
  • a peptide moiety can be tethered to the carrier molecule by a variety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage.
  • a peptide-based linkage can be a GFLG peptide.
  • the iRNA agents of the invention are particularly useful when targeted to the liver.
  • An iRNA agent can be targeted to the liver by incorporation of an RRMS containing a ligand that targets the liver.
  • a liver-targeting agent can be a lipophilic moiety.
  • Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues.
  • liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid,
  • dimethoxytrityl dimethoxytrityl, or phenoxazine.
  • An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL.
  • LDL low-density lipoprotein
  • Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.
  • a targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver.
  • a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other.
  • the targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose.
  • the targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine.
  • a mannose or mannose-6- phosphate targeting moiety can be used for macrophage targeting.
  • Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to the liver.
  • SA serum albumin
  • An iRNA agent targeted to the liver by an RRMS targeting moiety described herein can target a gene expressed in the liver.
  • the iRNA agent can target
  • the iRNA agent can target apoB-100, such as for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia; hypercholesterolemia; statin- resistant hypercholesterolemia; coronary artery disease (CAD); coronary heart disease (CHD); or atherosclerosis.
  • dyslipidemias e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia
  • hypercholesterolemia e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia
  • hypercholesterolemia CAD
  • coronary heart disease (CHD) coronary heart disease
  • atherosclerosis e.g., atherosclerosis.
  • the iRNA agent can target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR- Gamma Coactivator (PGC-1); Fructose- 1 ,6-bisphosphatase; glucose-6-phosphatase; glucoses- phosphate translocator; glucokinase inhibitory regulatory protein; or phosphoenolpyruvate carboxykinase (PEPCK), such as to inhibit hepatic glucose production in a mammal, such as a human, such as for the treatment of diabetes.
  • FKHR rhabdomyosarcoma
  • PLC-1 PPAR- Gamma Coactivator
  • Fructose- 1 ,6-bisphosphatase glucose-6-phosphatase
  • glucoses- phosphate translocator glucokinase inhibitory regulatory protein
  • PEPCK phosphoenolpyruvate carboxykin
  • an iRNA agent targeted to the liver can target Factor V, e.g., the Leiden Factor V allele, such as to reduce the tendency to form a blood clot.
  • An iRNA agent targeted to the liver can include a sequence which targets hepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H).
  • hepatitis virus e.g., Hepatitis A, B, C, D, E, F, G, or H.
  • an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B.
  • an iRNA agent can target the protein X (HBx) gene, for example.
  • Preferred ligands on RRMSs include folic acid, glucose, cholesterol, cholic acid, Vitamin E, Vitamin K, or Vitamin A.
  • halo refers to any radical of fluorine, chlorine, bromine or iodine.
  • alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms.
  • C1-C12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it.
  • haloalkyl refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S.
  • aralkyl refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “aralkyl” include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.
  • alkenyl refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups.
  • alkynyl refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl.
  • the sp 2 and sp 3 carbons may optionally serve as the point of attachment of the alkenyl and alkynyl groups, respectively.
  • alkoxy refers to an -O-alkyl radical.
  • aminoalkyl refers to an alkyl substituted with an amino
  • mercapto refers to an -SH radical.
  • thioalkoxy refers to an -S-alkyl radical.
  • alkylene refers to a divalent alkyl (i.e., -R-), e.g., -CH 2 -, -CH 2 CH 2 -, and - CH 2 CH 2 CH 2 -.
  • alkylenedioxo refers to a divalent species of the structure -O-R-O-, in which R represents an alkylene.
  • aryl refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted by a substituent.
  • aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl.
  • cycloalkyl as employed herein includes saturated cyclic, bicyclic, tricycliC j Or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom capable of substitution can be substituted by a substituent.
  • the cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon- carbon bond. Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and norbornyl.
  • heterocyclyl refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent.
  • the heterocyclyl groups herein described may also contain fused rings.
  • Fused rings are rings that share a common carbon-carbon bond.
  • heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.
  • heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent.
  • oxo refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.
  • acyl refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.
  • substituted refers to a group “substituted” on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group.
  • Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO 3 H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(0) n alkyl (where n is 0-2), S(0) n aryl (where n is 0-2), S(0) n heteroaryl (where n is 0-2), S(0) n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl
  • adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl refer to radicals of adenine, cytosine, guanine, thymine, and uracil.
  • an "unusual" nucleobase can include any one of the following:
  • the invention features an iRNA agent which can be asymmetrically modified as described herein.
  • the invention includes iRNA agents having asymmetrical modifications and another element described herein.
  • the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates an asymmetrical modification.
  • an iRNA agent described herein e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing
  • an iRNA agent which targets a gene described herein e.g., a gene active in the liver
  • iRNA agents of the invention can be asymmetrically modified.
  • An asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand.
  • An asymmetrical modification is a modification found on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification.
  • An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein.
  • an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue; modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway.
  • the designation of a modification as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting.
  • asymmetrical modification allows an iRNA agent to be optimized in view of the different or "asymmetrical" functions of the sense and antisense strands.
  • both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense strand .
  • some modifications e.g., targeting moieties
  • targeting moieties can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such modifications are preferably placed on the sense strand.
  • targeting moieties, especially bulky ones e.g.
  • an asymmetrical modification in which a phosphate of the backbone is substituted with S is present in the antisense strand, and a 2' modification, e.g., 2' OMe is present in the sense strand.
  • a targeting moiety can be present at either (or both) the 5' or 3' end of the sense strand of the iRNA agent.
  • a P of the backbone is replaced with S in the antisense strand
  • 2'OMe is present in the sense strand
  • a targeting moiety is added to either the 5' or 3' end of the sense strand of the iRNA agent.
  • an asymmetrically modified iRNA agent has a
  • Each strand can include one or more asymmetrical modifications.
  • one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA.
  • one strand, e.g., the sense strand can have a modification which targets the iRNA agent to a tissue
  • the other strand, e.g., the antisense strand has a modification which promotes hybridization with the target gene sequence.
  • both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well. E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand.
  • one strand has an asymmetrical 2' modification, e.g., a 2' OMe modification
  • the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification.
  • the antisense strand has an asymmetrical 2' OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa).
  • the RNAi agent will have asymmetrical 2'-0 alkyl, preferably, 2 '-OMe modifications on the sense strand and
  • asymmetrical backbone P modification preferably a phosphothioate modification in the antisense strand.
  • a particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region about 20-21, and preferably 19, subunits in length and one or two 3 ' overhangs of about 2 subunits in length.
  • Asymmetrical modifications are useful for promoting resistance to degradation by nucleases, e.g., endonucleases.
  • iRNA agents can include one or more asymmetrical
  • the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target.
  • Most if not all sites on a strand are vulnerable, to some degree, to degradation by endonucleases.
  • Particularly favored modifications include: 2' modification, e.g., provision of a 2' OMe moiety on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement of the U with a C5 amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2', 6', 7', or 8' position.
  • Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
  • Asymmetrical modification can be used to inhibit degradation by exonucleases.
  • Asymmetrical modifications can include those in which only one strand is modified as well as those in which both are modified.
  • the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target.
  • Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3 ' overhang, e.g., at the 3 ' terminus, and on the antisense strand, e.g., in a 3' overhang, e.g., at the 3' terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the
  • modifications which inhibit exonucleolytic degradation can be found herein.
  • Particularly favored modifications include: 2' modification, e.g., provision of a 2' OMe moiety in a 3 ' overhang, e.g., at the 3' terminus (3 ' terminus means at the 3 ' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P in a 3 ' overhang, e.g., at the 3 ' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and
  • modification of the backbone e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3 ' overhang, e.g., at the 3 ' terminus; modification with a 3' alkyl; modification with an abasic pyrolidine in a 3 ' overhang, e.g., at the 3 ' terminus; modification with naproxene, ibuprofen, or other moieties which inhibit degradation at the 3' terminus.
  • Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
  • Modifications e.g., those described herein, which affect targeting can be provided as asymmetrical modifications.
  • a biodistribution altering moiety e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand.
  • Targeting modifications which introduce moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive charge or one negative charge) can be placed on the sense strand.
  • Modifications e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical
  • iRNA agents have a defined structure, with a sense strand and an antisense strand, and in many cases short single strand overhangs, e.g., of 2 or 3 nucleotides are present at one or both 3 ' ends.
  • Asymmetrical modification can be used to optimize the activity of such a structure, e.g., by being placed selectively within the iRNA.
  • the end region of the iRNA agent defined by the 5' end of the sense strand and the 3 'end of the antisense strand is important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3 ' overhang.
  • asymmetrical modifications which result in one or more of the following are used: modifications of the 5' end of the sense strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5 ' exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a "tight" structure at this end of the molecule.
  • the end region of the iRNA agent defined by the 3' end of the sense strand and the 5 'end of the antisense strand is also important for function.
  • This region can include the terminal 2, 3, or 4 paired nucleotides and any 3 ' overhang.
  • Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an "open" structure at this end of the molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification of the antisense strand which inhibit kinase activation are avoided in preferred embodiments.
  • L sugars e.g., L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe
  • these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC;
  • modified sugars e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)
  • LNA's locked nucleic acids
  • HNA's hexose nucleic acids
  • CeNA's cyclohexene nucleic acids
  • nucleobase modifications e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines
  • these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand;
  • conjugate groups e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC.
  • L sugars e.g, L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe
  • modified sugars e.g., LNA's, HNA's and CeNA's
  • these modifications are preferably excluded from the 5' end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a "loose" structure in the 5' region, additionally, they may interfere with kinase activity;
  • nucleobase modifications e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines;
  • conjugate groups e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred.
  • the 5'-OH of the antisense strand should be kept free to promote activity.
  • modifications that promote nuclease resistance should be included at the 3 ' end, particularly in the 3 ' overhang.
  • the invention features a method of optimizing, e.g., stabilizing, an iRNA agent.
  • the method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity.
  • the decrease in activity can be less than a preselected level of decrease.
  • decrease in activity means a decrease of less than 5, 10, 20, 40, or 50 % activity, as compared with an otherwise similar iRNA lacking the introduced modification.
  • Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the required maintenance of activity.
  • the optimized property can be any property described herein and in particular the properties discussed in the section on asymmetrical modifications provided herein.
  • the modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein.
  • Particularly preferred asymmetric modifications are 2'-0 alkyl modifications, e.g., 2'-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate modifications, in the antisense sequence.
  • a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications.
  • a sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified.
  • the invention features an iRNA agent which can have a Z-X-Y architecture or structure such as those described herein and those described in copending, co- owned United States Provisional Application Serial No. 60/510,246 (Attorney Docket No. 14174-079P02), filed on October 9, 2003, which is hereby incorporated by reference, and in copending, co-owned United States Provisional Application Serial No. 60/510,318 (Attorney Docket No. 14174-079P03), filed on October 10, 2003, which is hereby incorporated by reference.
  • the invention includes iRNA agents having a Z-X-Y structure and another element described herein.
  • the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a Z-X-Y architecture.
  • the invention provides an iRNA agent having a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region:
  • Z— X— Y It may be desirable to modify subunits in one or both of Zand/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X.
  • the Z region typically includes a terminus of an iRNA agent.
  • the length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some embodiments self associate, e.g., to form a loop structure.
  • Such structures can be formed by the end of a strand looping back and forming an intrastrand duplex.
  • 2, 3, 4, 5 or more intra-strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand.
  • a typical embodiment of a Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein.
  • the Z region can thus be or include a 3 ' or 5 ' terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3- overhang.
  • Chiral P X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.)
  • Other preferred Z region subunit modifications can include: 3 '-OR, 3'SR, 2'-OMe, 3'-OMe, and 2 ⁇
  • the X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand.
  • the length of the X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits.
  • Particularly preferred region X's will include 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengths are described elsewhere herein and can be used.
  • Typical X region subunits include 2'-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phophorothioate or non-phosphate bonds are absent.
  • modifications preferred in the X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines.
  • Some embodiments have 4 or more consecutive 2 ⁇ subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2 ⁇ subunits.
  • the Y region will generally conform to the the parameters set out for the Z regions. However, the X and Z regions need not be the same, different types and numbers of
  • the iRNA agent will have a Y and/or Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe or other alkyl; and an X region that includes at least four consecutive ribonucleoside subunits in which the 2'-OH remains unsubstituted.
  • the subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage.
  • the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe; and an X region that includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2' -OH remains unsubstituted.
  • the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in particular it is preferred that consecutive modified linkages be avoided.
  • not all of the subunit linkages of the iRNA agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4.
  • Particulary preferred iRNA agents will not have four or more consecutive subunits, e.g., 2'-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.
  • each of the nucleoside subunit linkages in X will be phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized.
  • the Y and/or Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized.
  • the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4.
  • Particulary preferred X regions will not have four or more consecutive subunits, e.g., 2'-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.
  • modified linkages e.g. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.
  • Y and /or Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications of the subunit linkages.
  • region X or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2' moieties.
  • region X or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages.
  • one or more phosphorothioate linkages are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., a 2'-0-alkyl moiety.
  • any adjacent 2'-0-alkyl moieties in the Y and/or Z are connected by a linkage other than a a phosphorothioate linkage.
  • each of Y and/or Z independently has only one
  • phosphorothioate linkage between adjacent subunits e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides.
  • adjacent subunits e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides.
  • second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.
  • each of Y and/orZ independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides, are be connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.
  • one of the above recited limitation on adjacent subunits in Y and or Z is combined with a limitation on the subunits in X.
  • one or more phosphorothioate linkages are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., a 2'-0-alkyl moiety.
  • any adjacent 2'-0-alkyl moieties in the Y and/or Z are connected by a linkage other than a phosporothioate linkage.
  • the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2' moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages.
  • a Y and/or Z region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein.
  • modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety.
  • more than 1 of such subunits can be present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.
  • the frequency of the modification will differ between Y and /or Z and X, e.g., the modification will be present one of Y and/or Z or X and absent in the other.
  • An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein.
  • modifications can be chosen, independently, from any modification desribed herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety.
  • more than 1 of such subunits can b present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.
  • An RRMS (described elswhere herein) can be introduced at one or more points in one or both strands of a double-stranded iRNA agent.
  • An RRMS can be placed in a Y and/or Z region, at or near (within 1, 2, or 3 positions) of the 3 ' or 5' end of the sense strand or at near (within 2 or 3 positions of) the 3 ' end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end of the antisense strand.
  • An RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical for antisense binding to the target.
  • the invention features an iRNA agent which can have differential modification of terminal duplex stability (DMTDS).
  • DMTDS differential modification of terminal duplex stability
  • the invention includes iRNA agents having DMTDS and another element described herein.
  • the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS.
  • an iRNA agent described herein e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing
  • an iRNA agent which targets a gene described herein e.g., a gene active in the liver
  • an iRNA agent having an architecture or structure described herein an
  • iRNA agents can be optimized by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5' end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5' end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5 'end . While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5' end of the antisense strand.
  • iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex association), in the region of the 3 ' end of the antisense strand duplex. This can be
  • Modifications which increase the tendency of the 5' end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3' end of the duplex to dissociate.
  • modifications which decrease the tendency of the 3' end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5' end of the duplex to dissociate.
  • Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used).
  • dissociation or melting e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used.
  • A:U is preferred over G:C;
  • G:U is preferred over G:C;
  • mismatches e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings;
  • a typical ds iR A agent can be diagrammed as follows: s 5' Ri Ni N 2 N 3 N 4 N 5
  • S indicates the sense strand
  • AS indicates antisense strand
  • Ri indicates an optional (and nonpreferred) 5' sense strand overhang
  • R 2 indicates an optional (though preferred) 3' sense overhang
  • R3 indicates an optional (though preferred) 3 ' antisense sense overhang
  • R4 indicates an optional (and nonpreferred) 5' antisense overhang
  • N indicates subunits
  • [N] indicates that additional subunit pairs may be present
  • P x indicates a paring of sense N x and antisense N x . Overhangs are not shown in the P diagram.
  • a 3 ' AS overhang corresponds to region Z
  • the duplex region corresponds to region X
  • the 3 ' S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.)
  • pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5' end of the AS strand.
  • the terminal pair (the most 5' pair in terms of the AS strand) is designated as P_i, and the subsequent pairing positions (going in the 3 ' direction in terms of the AS strand) in the duplex are designated, P -2 , P-3, P_ 4 , P-5, and so on.
  • the preferred region in which to modify to modulate duplex formation is at P_5 through P_i, more preferably P_ 4 through P_i , more preferably P-3 through P_i.
  • Modification at P_ 1, is particularly preferred, alone or with modification(s) other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of:
  • mismatched pairs e.g., non-canonical or other than canonical pairings or pairings which include a universal base.
  • the change in subunit needed to achieve a pairing which promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand.
  • the at least 2, or 3, of the pairs in P_i, through P_ 4 are pairs which promote disociation.
  • the at least 2, or 3, of the pairs in P_i, through P_ 4 are A:U.
  • the at least 2, or 3, of the pairs in P_i, through P_ 4 are G:U.
  • the at least 2, or 3, of the pairs in P_i, through P_ 4 are I:C.
  • the at least 2, or 3, of the pairs in P_i, through P_ 4 are mismatched pairs, e.g., non-canonical or other than canonical pairings pairings.
  • the at least 2, or 3, of the pairs in P_i, through P_ 4 are pairings which include a universal base.
  • Subunit pairs can be ranked on the basis of their propensity to promote stability and inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used).
  • dissociation or melting e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used.
  • G:C is preferred over A:U
  • G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G guanadinium-G-clamp:G is preferred over C:G
  • sugar modifications e.g., 2' modifications, e.g., 2'F, ENA, or LNA, which enhance binding are preferred over non-modified moieties and can be present on one or both strands to enhance stability of the duplex.
  • pairings which increase the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 3 ' end of the AS strand.
  • the terminal pair (the most 3 ' pair in terms of the AS strand) is designated as Pi, and the subsequent pairing positions (going in the 5' direction in terms of the AS strand) in the duplex are designated, P2, P3, P 4 , P5, and so on.
  • the preferred region in which to modify to modulate duplex formation is at P 5 through Pi, more preferably P 4 through Pi , more preferably P3 through Pi.
  • Modification at Pi is particularly preferred, alone or with mdification(s) at other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of the recited regions be chosen independently from the group of:

Abstract

The invention relates to compositions and methods of modulating the status, activity or expression of long intervening non-coding RNA transcript (lncRNA) targets.

Description

METHODS AND COMPOSITIONS FOR THE REGULATION OF RNA
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This Patent Application claims the priority of U.S. Patent Application No. 61/370,414 filed on 3 August 2010, entitled, "Methods and Compositions for the Regulation of RNA", the contents and teachings of which are hereby incorporated by reference in their entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The instant application contains a "lengthy" Sequence Listing which has been submitted via CD-R in lieu of a printed paper copy, and is hereby incorporated by reference in its entirety. This contains a sequence listing text file as part of the originally filed subject matter as follows: File name: 20021PCT.TXT; File size: 190,869,504 bytes; Date created: 19 May 2011. These CD-Rs are labeled "CRF," "Copy 1," "Copy 2," and "Copy 3" respectively, and each contain only one identical file, as identified immediately above.
REFERENCE TO LENGTHY TABLE
[0003] The specification includes a lengthy Table 1. Lengthy Table 1 has been submitted via EFS-Web in electronic format as follows: File name: MLTM-001PCT_lncRNA_Table_l.txt, Date created: May 25, 201 1; File size: 12,096,042 bytes and is incorporated herein by reference in its entirety. Please refer to the end of the specification for access instructions.
FIELD OF THE INVENTION
[0004] The invention relates to the regulation of non-coding RNAs, specifically long intervening non-coding RNA (IncRNA) genes and transcripts.
BACKGROUND OF THE INVENTION
[0005] Non-coding RNA (ncRNA) genes produce functional RNA molecules rather than encoding proteins. However ncRNA genes have been poorly characterized and only recently the topic of serious inquiry. Several ncRNA genes have recently been identified and these appear to have diverse roles in cellular regulation and gene expression as structural, catalytic and regulatory molecules (See Eddy S. Curr Opin Genet Devel, 9: 695-699, 1999; Eddy, S. Nature Reviews, Genetics, 2: 919-929, 2001; and Mattick, J., et al., Mol. Biol. & Evolution, vol. 18, no. 9, 16: 1611-1630, 2001). In US Patent, 6,891,031, Frazer et al, disclose certain conserved noncoding sequences (CNS) that regulate the cytokine gene expression.
[0006] The most prominent and well-studied example of a functional ncRNA in the literature to date is the ncRNA, XIST (Plath, et al, Science, 300; 131-135; 2003). In 2007, investigating the HOX cluster, Rinn and colleages identified the gene known as HOTAIR, which represses transcription in trans across the HOXD cluster, suggesting that ncRNA may play a critical role in chromosomal demarcation and chromatin states that affect cancer metastasis (Rinn, et al, Cell, 129(7); 131 1-1323; 2007; Gupta, R., et al, Nature Letters, 464, 1071; 2010; See also Koziol and Rinn, Curr. Opinion in Gen. Dev., 20, 142-148; 2010).
[0007] Recently, efforts by several groups applying a variety of selection protocols have identified a set of large non-coding RNA genes. Termed "long intervening" and/or "long intergenic" RNAs, these genes were collectively referred to as lincRNAs. These studies focused on the identification of lincRNA genes by investigating chromatin state maps and specific methylation patterns. (Guttman, M., et al, Nature, 458, 223-227; 2009; Kalil, A., et al, Proc. Natl. Acad. Sci., 106, 11667-672; 2009). Follow-on studies of mouse transcriptomes revealed that lincRNAs contain features of normal coding genes including promoters and a multi-exonic structure (Guttman, M., et al., Nature Biotech., 1-8, 2010). While this work was facilitated by the creation of whole transcriptome libraries of mouse and human tissues (Okazaki, Y., et al., Nature, 420: 563-573, 2002), much is still unknown about the large category of non-coding RNA genes, their transcripts and role in gene regulation and cell biology.
[0008] Other categories of ncRNAs including natural antisense transcripts (NATs) and pseudogenes have been shown to have coding-independent functions (Poliseno, L., et al, Nature, 465: 1033-1038, 2010); and NATs have been investigated for their role in the development and role in the nervous system and as potential drug targets (Wahlestedt, C, Drug Discovery Today, 1 1 : 503-508; 2006; Faghihi, M., and Wahlstedt, C, Genome Biology, 7:R38, 2006; St. Laurent, G., and Wahlstedt, C, TRENDS in Neuro., 30: 612-621; Faghihi, M., et al, Nat. Med.,
14(7):723-30, 2008; Faghihi, M., and Wahlstedt, C, Nat. Rev. Mol. Cell Biol., 10(9):637-43, 2009; St. Laurent, G., et al, Neuroscience Letters, 466: 81-8, 2009; Faghihi, M., et al, Genome Biology, 11 :R56, 2010; see also PCT/US2006/062672).
[0009] There has been, however, much difficulty identifying bona fide ncRNA genes given their lack of open reading frames and no scientific consensus around rules or metrics for qualifying a transcript as a true lincRNA based on sequence features (Babak, T, et al, BMC Genomics, 6, 104; 2005; Carninci et al, Science 309 (5740), 1559-1563, 2005; Ponjavic, J., Genome Research, 17: 556-65; 2007; Marques, A., et al, Genome Biology, 10; R124; 2009; Louro, R., et al, Genomics, 93:291-298, 2009). [0010] Consequently, there remains a need for further investigation of the noncoding transcriptome of the mammalian cell to identify ncRNA genes and determine their function in biology and ultimately to identify methods and compositions for their effective manipulation for therapeutic outcomes.
SUMMARY OF THE INVENTION
[0011] Described herein are compositions and methods useful in the control, regulation, exploitation and study of non-coding RNA (ncRNA) transcripts including ncRNA genes, particularly long intervening (which includes both intronic and intergenic) RNAs (IncRNAs) and the like in cells in vitro, in vivo, ex vivo and in ovo. Also described are compositions and methods for the diagnosis, prevention, amelioration and/or treatment of pathological conditions and diseases involving the status, activity, or expression of non-coding RNAs, particularly lncRNA genes and most particularly those lncRNA genes and nearest neighbor coding genes identified herein.
[0012] The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Described herein are compositions and methods of modulation the status, activity, or expression of long intervening (which includes both intronic and intergenic) non-coding RNAs (IncRNAs) in a cell, tissue or organism. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by or modulated by the regulatory, structural, catalytic or signaling properties of a lncRNA. Further disclosed are diagnostic methods, kits and assays which are designed to utilize the cellular pathways and systems associated with lncRNA targets.
[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the IncRNA-directed agents (LDAs) and methods featured in the invention, suitable methods and materials are described below.
I. lncRNA targets
[0015] Targets of the present invention include long intervening (intronic and intergenic) non-coding RNAs, or "IncRNAs", also known in the art as macroRNAs and efference RNAs (eRNAs). As used herein, the term "lncRNA", or "long intervening non-coding RNA" refers broadly to the targets of the present invention and include the "IncRNA gene", as well as the resultant "IncRNA transcript." To the extent that the IncRNA transcript acts as an antisense molecule (whether in cis or trans to effect concordant or discordant regulation) to a second transcript (whether RNA or DNA), the family of IncRNA targets envisioned by the present inventors also includes NATs (Natural Antisense Transcripts). Further, pseudogenes, whether arising from transposition or duplication, are also considered to fall within the broader family of IncRNA targets of the present invention.
[0016] "IncRNA genes" of the present invention are processed to produce "IncRNA transcripts" and these transcripts may be transcribed from either strand of the chromosomal DNA. Unless otherwise noted, the term "IncRNA" refers broadly to the IncRNA gene, as well as the resultant IncRNA transcript. IncRNA genes may be as small as lkb (kilobase) or as large as lOOkb (kilobases) while IncRNA transcripts may range in size from 200 nucleotides to 20kb.
[0017] IncRNA transcripts may be at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, at least 5000 nucleotides, at least 10,000 nucleotides or at least 20,000 nucleotides, and range from 250-300 nucleotides, 300- 400 nucleotides, 400-500 nucleotides, 500-600 nucleotides, 600-700 nucleotides, 700-800 nucleotides, 800-900 nucleotides, 900-1000 nucleotides, 1000-5000 nucleotides, 5000-10,000 nucleotides, or 10,000-20,000 nucleotides in length.
[0018] As used herein, the term "IncRNA gene" refers to the IncRNA which is encoded within the genome or in a genomic construct (whether natural or synthetic) and has at least one feature of a coding gene selected from the group including, but not limited to, (i) a promoter or promoter-like feature such as one or more proximal regulatory elements; (ii) one or more exons; (iii) a polyA signature and (iv) which encodes a transcribed RNA, i.e., a IncRNA transcript. Endogenous IncRNA genes, e.g., those encoded within or engineered to be encoded by a host cell genome, are characterized by their intervening genomic location. This means that endogenous or wildtype IncRNAs may be intronic or intergenic.
[0019] "Intronic IncRNAs" are those found to be encoded substantially within an intron of a gene.
[0020] "Intergenic IncRNAs" are those found to be encoded between two different genes. As used herein, the term "IncRNA transcript", refers to an RNA transcript encoded by a IncRNA gene and which is (i) at least 200 nucleotides in length and (ii) does not encode a mature or complete protein product. IncRNA transcripts may encode peptides of 50 amino acids or less. It should be understood that IncRNA transcripts may be synthesized as IncRNA transcript variants, which may be engineered to encode peptides or proteins. [0021] Other types of large non-coding RNA transcripts found in cells such as primary microRNAs (pri-miRs) which are processed to produce micro-RNAs or small RNA species are not targets of the present invention.
[0022] "Intervening" when used in the context of IncRNAs means intronic or intergenic.
[0023] Representative IncRNA genes and transcript targets are listed in Table 2. IncRNA genes were identified from the Ensembl database (www.ensembl.org) and their respective RNA transcripts were extracted. Table 2 lists the Ensembl gene identifier of each IncRNA gene, (prefix ENSG), each IncRNA transcript (prefix ENST), in addition to the human chromosomal location of each gene (column 1, designated "C").
[0024] Also reported in Table 2 are the results of nearest neighbor analyses of the IncRNA genes. In the columns labeled 5' Gene and 3 ' Gene are reported the gene symbol of the gene which contains the nearest upstream exon of a coding gene and the nearest downstream exon of a protein coding gene, respectively. The table lists the gene symbol of the gene from which the upstream or downstream exon is derived as well as the NCBI sequence references for each gene (5' RefSeq and 3'RefSeq).
[0025] The naming convention used follows that of the annotation file, e.g.,
"Homo_sapiens.GRCh37.58.gif, i.e., annotation version 58 from the Ensembl database. The sequence as well as other characteristics of each upstream and downstream gene identified may be identified by searching the Ensembl database or the NCBI database using the unique identifiers reported in Table 2.
[0026] The results of nearest neighbor analysis (See Example 1) revealed that IncRNA transcripts are found either within an intron of a coding gene or encoded between two coding genes. The nature of each of the identified IncRNA genes is reported in Table 2 as INT
(meaning intronic) or ING (meaning intergenic). Where the same gene name is listed in both the upstream (5') and downstream (3 ') Gene columns, it is to be concluded that the IncRNA gene is intronic, i.e., found encoded within an intron of a coding gene.
[0027] Given the proximity of the IncRNA genes and their transcripts to the coding genes identified, it is expected that the lncRNA-directed agents (LDAs) of the present invention which are directed to a IncRNA gene or transcript could be designed to modulate, regulate or otherwise alter or change the expression patterns or activity of one or more of the upstream or downstream protein coded genes.
[0028] Likewise, by targeting a nearest neighbor coding gene, the IncRNA expression pattern or activity can be altered. Table 2
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[0029] Protein coding genes found upstream of IncRNA genes or transcripts may be targeted by any of the methods disclosed herein. These genes include, but are not limited to, 2',3'-cyclic nucleotide 3' phosphodiesterase; 3-phosphoinositide dependent protein kinase-1 ; 5'-nucleotidase domain containing 2; 5 '-nucleotidase, cytosolic II; A kinase (PRKA) anchor protein 1 1; A kinase (PRKA) anchor protein 8-like; abhydrolase domain containing 6; acetyl-CoA acyltransferase 2; acetyl-CoA carboxylase alpha; activating transcription factor 7 interacting protein 2; acyl-CoA oxidase 1, palmitoyl; acyl-CoA synthetase bubblegum family member 2; acyl-CoA synthetase long-chain family member 3; acyl-CoA synthetase medium-chain family member 1 ; acyl-CoA synthetase short-chain family member 1 ; acyl-CoA thioesterase 12; acyl-CoA thioesterase 13; acyl-malonyl condensing enzyme 1 ; acylphosphatase 2, muscle type; ADAM metallopeptidase domain 10; ADAM metallopeptidase domain 19 (meltrin beta); ADAM metallopeptidase with thrombospondin type 1 motif, 2; adaptor-related protein complex 1, gamma 2 subunit; adaptor- related protein complex 2, beta 1 subunit; adaptor-related protein complex 2, sigma 1 subunit; ADP-ribosylation factor 6; ADP-ribosylation factor guanine nucleotide-exchange factor l(brefeldin A-inhibited); ADP-ribosylation factor interacting protein 1; ADP-ribosylation factorlike 13 A; ADP-ribosylation factor-like 17 A; ADP-ribosylation factor-like 9; adrenergic, alpha- 2C-, receptor; AF4/FMR2 family, member 1; aldehyde dehydrogenase 1 family, member A2; aldehyde dehydrogenase 1 family, member A3; aldo-keto reductase family 1, member CI (dihydrodiol dehydrogenase 1 ; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase); alkB, alkylation repair homolog 3 (E. coli); alkylglycerone phosphate synthase; alpha 1,4- galactosyltransferase; amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein; angiopoietin 2; ankyrin 3, node of Ranvier (ankyrin G); ankyrin repeat and SOCS box-containing 3; ankyrin repeat and SOCS box-containing 7; ankyrin repeat and sterile alpha motif domain containing IB; ankyrin repeat domain 11 ; annexin A4; antigen p97
(melanoma associated) identified by monoclonal antibodies 133.2 and 96.5; apolipoprotein D; ArfGAP with GTPase domain, ankyrin repeat and PH domain 4; ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 1 ; ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 3; ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 ; argininosuccinate synthase 1; ARP3 actin-related protein 3 homolog C (yeast); arrestin domain containing 1; aryl hydrocarbon receptor nuclear translocator-like 2; ASF1 anti-silencing function 1 homolog B (S. cerevisiae); asparagine synthetase (glutamine-hydrolyzing); asparagine-linked glycosylation 8, alpha-1,3- glucosyltransferase homolog (S. cerevisiae); aspartate beta-hydroxylase; aspartic peptidase, retroviral-like 1; AT rich interactive domain 1A (SWI-like); AT rich interactive domain 5B (MRFl-like); ATP binding domain 4; ATPase, Ca++ transporting, cardiac muscle, fast twitch 1; ATPase, Ca++ transporting, type 2C, member 2; ATPase, class VI, type 1 1A; ATPase, H+ transporting VO subunit e2; ATPase, H+ transporting, lysosomal 3 IkDa, VI subunit E2; ATPase, H+ transporting, lysosomal 42kDa, VI subunit CI ; ATPase, H+ transporting, lysosomal 70kDa, VI subunit A; ATPase, Na+/K+ transporting, beta 3 polypeptide; ATP-binding cassette, subfamily B (MDR/TAP), member 4; B double prime 1 , subunit of RNA polymerase III transcription initiation factor IIIB; BAI1 -associated protein 2-like 1; Bardet-Biedl syndrome 4; BARX homeobox 2; B-cell CLL/lymphoma 7 A; B-cell translocation gene 1, anti-proliferative; BCL6 corepressor; benzodiazapine receptor (peripheral) associated protein 1 ; bestrophin 3; beta- 1,3-glucuronyltransferase 1 (glucuronosyltransferase P); beta-2-microglobulin; BMP2 inducible kinase-like; bolA homolog 3 (E. coli); bone marrow stromal cell antigen 2; bone morphogenetic protein 8a; brain and acute leukemia, cytoplasmic; brain and reproductive organ-expressed (TNFRSF1A modulator); breast carcinoma amplified sequence 3; brevican; BRF2, subunit of RNA polymerase III transcription initiation factor, BRF 1 -like; BRI3 binding protein;
bromodomain PHD finger transcription factor; BTB and CNC homology 1 , basic leucine zipper transcription factor 2; butyrophilin, subfamily 2, member Al; C2 calcium-dependent domain containing 4B; cadherin 10, type 2 (T2-cadherin); cadherin 11, type 2, OB-cadherin (osteoblast); cadherin 18, type 2; cadherin 20, type 2; cadherin 5, type 2 (vascular endothelium); cadherin 9, type 2 (Tl -cadherin); calbindin 1, 28kDa; calcineurin binding protein 1; calcium channel, voltage-dependent, L type, alpha 1C subunit; calcium homeostasis endoplasmic reticulum protein; caldesmon 1 ; cAMP responsive element binding protein 5; cancer susceptibility candidate 5; cancer/testis antigen family 47, member Al; CAP, adenylate cyclase-associated protein, 2 (yeast); caprin family member 2; carbohydrate (chondroitin 4) sulfotransferase 1 1; carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 14; carbonic anhydrase VIII;
carboxylesterase 7; carboxylesterase 7; carboxypeptidase A4; carboxypeptidase A5;
carboxypeptidase E; carnitine palmitoyltransferase 1A (liver); carnosine dipeptidase 1
(metallopeptidase M20 family); casein kinase 1, delta; CASK interacting protein 1 ; CASP2 and RIPKl domain containing adaptor with death domain; caspase recruitment domain family, member 8; catalase; catenin (cadherin-associated protein), beta 1, 88kDa; cathepsin B; cathepsin L2; CCCTC-binding factor (zinc finger protein)-like; CD320 molecule; CD3e molecule, epsilon (CD3-TCR complex); CD44 molecule (Indian blood group); CD6 molecule; CD97 molecule; CDC-like kinase 4; cell adhesion molecule 1 ; cell adhesion molecule 1 ; cell division cycle 27 homolog (S. cerevisiae); cell division cycle 73, Pafl/RNA polymerase II complex component, homolog (S. cerevisiae); cell division cycle and apoptosis regulator 1 ; cell division cycle associated 4; cellular repressor of ElA-stimulated genes 1; CGRP receptor component;
checkpoint with forkhead and ring finger domains; chemokine (C-C motif) ligand 15; chemokine (C-C motif) ligand 4-like 1; chemokine (C-C motif) ligand 5; chemokine (C-X-C motif) ligand 2; chemokine (C-X-C motif) receptor 2; chemokine (C-X-C motif) receptor 3; chemokine (C-X- C motif) receptor 5; chemokine-like receptor 1 ; cholinergic receptor, nicotinic, alpha 4; chondrolectin; chromobox homolog 4 (Pc class homolog, Drosophila); chromobox homolog 7; chromodomain helicase DNA binding protein 4; chromosome 1 open reading frame 124;
chromosome 1 open reading frame 151 ; chromosome 1 open reading frame 43; chromosome 1 open reading frame 86; chromosome 10 open reading frame 114; chromosome 10 open reading frame 71; chromosome 1 1 open reading frame 2; chromosome 1 1 open reading frame 34;
chromosome 1 1 open reading frame 61; chromosome 12 open reading frame 33; chromosome 12 open reading frame 48; chromosome 14 open reading frame 147; chromosome 14 open reading frame 153; chromosome 14 open reading frame 159; chromosome 14 open reading frame 166B; chromosome 14 open reading frame 17; chromosome 14 open reading frame 182; chromosome 14 open reading frame 4; chromosome 14 open reading frame 49; chromosome 15 open reading frame 44; chromosome 15 open reading frame 53; chromosome 16 open reading frame 46; chromosome 16 open reading frame 47; chromosome 16 open reading frame 53; chromosome 16 open reading frame 54; chromosome 17 open reading frame 108; chromosome 17 open reading frame 65; chromosome 17 open reading frame 95; chromosome 18 open reading frame 62; chromosome 19 open reading frame 21; chromosome 19 open reading frame 69; chromosome 2 open reading frame 65; chromosome 20 open reading frame 112; chromosome 20 open reading frame 1 17; chromosome 20 open reading frame 96; chromosome 21 open reading frame 57; chromosome 22 open reading frame 39; chromosome 22 open reading frame 40; chromosome 3 open reading frame 1 ; chromosome 3 open reading frame 23 ; chromosome 4 open reading frame 19; chromosome 4 open reading frame 34; chromosome 4 open reading frame 41 ; chromosome 5 open reading frame 25; chromosome 6 open reading frame 223; chromosome 7 open reading frame 42; chromosome 7 open reading frame 50; chromosome 7 open reading frame 60;
chromosome 8 open reading frame 37; chromosome 8 open reading frame 38; chromosome 8 open reading frame 42; chromosome 8 open reading frame 45; chromosome 8 open reading frame 56; chromosome 8 open reading frame 56; chromosome 8 open reading frame 83;
chromosome 9 open reading frame 3; chromosome X open reading frame 23; CKLF-like MARVEL transmembrane domain containing 4; cleavage and polyadenylation specific factor 4- like; cleavage stimulation factor, 3' pre-RNA, subunit 3, 77kDa; CMT1A duplicated region transcript 15; CMT1A duplicated region transcript 4; coagulation factor C homolog, cochlin (Limulus polyphemus); coatomer protein complex, subunit epsilon; COBW domain containing 5; coiled-coil domain containing 102B; coiled-coil domain containing 54; coiled-coil domain containing 57; coiled-coil domain containing 60; coiled-coil domain containing 8; coiled-coil domain containing 85 C; coiled-coil-helix-coiled-coil-helix domain containing 1 ; collagen, type XXIII, alpha 1; colony stimulating factor 3 (granulocyte); COMM domain containing 10;
congenital dyserythropoietic anemia, type I; consortin, connexin sorting protein; contactin 5; contactin associated protein-like 2; contactin associated protein-like 3B; contactin associated protein-like 4; copper metabolism (Murrl) domain containing 1 ; core-binding factor, runt domain, alpha subunit 2; translocated to, 3; corticotropin releasing hormone; CREB regulated transcription coactivator 3; Crm, cramped-like (Drosophila); CTD (carboxy -terminal domain, R A polymerase II, polypeptide A) small phosphatase 2; CTP synthase; C-type lectin domain family 4, member M; CUE domain containing 2; CUGBP, Elav-like family member 4; cullin- associated and neddylation-dissociated 1; cyclin-dependent kinase 1 1 A; cyclin-dependent kinase 6; cylicin, basic protein of sperm head cytoskeleton 2; cysteine dioxygenase, type I; cysteine-rich protein 3; cysteine-rich secretory protein LCCL domain containing 1; cysteinyl-tRNA synthetase; cytochrome b5 domain containing 1 ; cytochrome c oxidase subunit Va; cytohesin 2; cytohesin 4; cytokine induced apoptosis inhibitor 1; cytoplasmic FMR1 interacting protein 2; cytoplasmic polyadenylation element binding protein 1; cytoskeleton associated protein 5; D- amino-acid oxidase; dapper, antagonist of beta-catenin, homolog 3 (Xenopus laevis); DAZ interacting protein 1 ; DEAD (Asp-Glu-Ala-As) box polypeptide 19B; DEAD (Asp-Glu-Ala- Asp) box polypeptide 53; DEAH (Asp-Glu-Ala-His) box polypeptide 16; decapping enzyme, scavenger; dedicator of cytokinesis 1; de-etiolated homolog 1 (Arabidopsis); defective in sister chromatid cohesion 1 homolog (S. cerevisiae); dehydrogenase/reductase (SDR family) member 12; deiodinase, iodothyronine, type II; deleted in bladder cancer 1 ; delta-like 1 homolog
(Drosophila); dermatan sulfate epimerase-like; desmoglein 2; developmental pluripotency associated 4; diaphanous homolog 1 (Drosophila); dicer 1, ribonuclease type III; dickkopf-like 1 (soggy); differentially expressed in FDCP 8 homolog (mouse); dihydrolipoamide branched chain transacylase E2; dihydropyrimidinase-like 3; dipeptidyl-peptidase 9; DIS3 mitotic control homolog (S. cerevisiae)-like; discs, large (Drosophila) homolog-associated protein 1 ; disrupted in renal carcinoma 2; DnaJ (Hsp40) homolog, subfamily C, member 5 gamma; dopey family member 1; DOT 1 -like, histone H3 methyltransferase (S. cerevisiae); doublecortin-like kinase 1; doublesex and mab-3 related transcription factor 3; Down syndrome cell adhesion molecule like 1; dpy-19-like 1 (C. elegans); drebrin 1; dual specificity phosphatase 10; dual specificity phosphatase 4; dynactin 5 (p25); dynactin 6; dynein, cytoplasmic 1, light intermediate chain 2; dynein, light chain, LC8-type 1 ; dynein, light chain, roadblock-type 2; dystroglycan 1
(dystrophin-associated glycoprotein 1); E2F transcription factor 2; E74-like factor 1 (ets domain transcription factor); early B-cell factor 1 ; early B-cell factor 3; echinoderm microtubule associated protein like 2; echinoderm microtubule associated protein like 5; echinoderm microtubule associated protein like 6; ECSIT homolog (Drosophila); egf-like module containing, mucin-like, hormone receptor-like 2; EH-domain containing 4; elastin microfibril interfacer 3; ELKS/RAB6-interacting/CAST family member 1 ; elongation factor 1 homolog (S. cerevisiae); elongation factor Tu GTP binding domain containing 1 ; embryonic ectoderm development; endonuclease domain containing 1 ; endoplasmic reticulum-golgi intermediate compartment (ERGIC) 1 ; endosulfine alpha; endothelial cell adhesion molecule; enhancer of rudimentary homolog (Drosophila); enolase superfamily member 1; erythrocyte membrane protein band 4.1 like 5; eukaryotic translation initiation factor 4 gamma, 2; eukaryotic translation initiation factor 4B; even-skipped homeobox 1 ; exocyst complex component 4; exosome component 3; exportin 6; family with sequence similarity 105, member A; family with sequence similarity 111, member B; family with sequence similarity 113, member B; family with sequence similarity 125, member B; family with sequence similarity 126, member A; family with sequence similarity 156, member B; family with sequence similarity 158, member A; family with sequence similarity 178, member B; family with sequence similarity 179, member B; family with sequence similarity 186, member B; family with sequence similarity 19 (chemokine (C-C motif)-like), member A2; family with sequence similarity 27, member B; family with sequence similarity 46, member C; family with sequence similarity 59, member A; family with sequence similarity 65, member B; family with sequence similarity 66, member A; family with sequence similarity 72, member D; family with sequence similarity 84, member B; family with sequence similarity 86, member C; family with sequence similarity 90, member Al ; family with sequence similarity 92, member B; farnesyltransferase, CAAX box, beta; FAT tumor suppressor homolog 2 (Drosophila); fatty acid amide hydrolase 2; fatty acid binding protein 6, ileal; fatty acyl CoA reductase 2; FBJ murine osteosarcoma viral oncogene homolog B; F-box and leucine-rich repeat protein 17; F-box and WD repeat domain containing 8; F-box protein 33; F-box protein 4; F-box protein 41 ; F-box protein 7; FERM domain containing 4A; FERM domain containing 6; FERM domain containing 8; ferredoxin-fold anticodon binding domain containing 1 ; FGFR1 oncogene partner 2; fibroblast growth factor 3 (murine mammary tumor virus integration site (v-int-2) oncogene homolog); fibroblast growth factor 9 (glia-activating factor); fibronectin type III domain containing 3B; fidgetin-like 1 ; FK506 binding protein IB, 12.6 kDa; fms-related tyrosine kinase 3; forkhead box F2; forkhead box II ; forkhead box Jl ; forkhead box LI; formin-like 1 ; formyl peptide receptor 3; four and a half LIM domains 2; FRAS l related extracellular matrix 3; frizzled homolog 3 (Drosophila); frizzled homolog 4 (Drosophila); FSHD region gene 2 family, member C;
fucosyltransferase 3 (galactoside 3(4)-L-fucosyltransferase, Lewis blood group);
fumarylacetoacetate hydrolase (fumarylacetoacetase); furin (paired basic amino acid cleaving enzyme); FY oncogene related to SRC, FGR, YES; G kinase anchoring protein 1; G protein regulated inducer of neurite outgrowth 1; G protein-coupled estrogen receptor 1; G protein- coupled receptor 107; G protein-coupled receptor 176; G protein-coupled receptor 180; G protein-coupled receptor 20; G protein-coupled receptor 81; GA binding protein transcription factor, beta subunit 1 ; galanin receptor 1; gamma-glutamyltransferase light chain 2; gap junction protein, delta 2, 36kDa; GATA zinc finger domain containing 2B; GDP-mannose 4,6- dehydratase; gelsolin; general transcription factor IIA, 1, 19/37kDa; general transcription factor IIH, polypeptide 2, 44kDa; general transcription factor IIH, polypeptide 2C; general transcription factor IIH, polypeptide 5; germ cell associated 1 ; GIPC PDZ domain containing family, member 1; glial fibrillary acidic protein; glioblastoma amplified sequence; glucan (1,4-alpha-), branching enzyme 1 ; glucocorticoid deficiency 3; glutamate receptor, ionotropic, delta 1 ; glutamate receptor, ionotropic, kainate 4; glutamate receptor, ionotropic, kainate 5; glutamate receptor, metabotropic 7; glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2); glutaminase; glyceronephosphate O-acyltransferase; glycine amidinotransferase (L- arginine:glycine amidinotransferase); glycoprotein VI (platelet); golgi membrane protein 1 ; golgi-associated PDZ and coiled-coil motif containing; golgi-associated, gamma adaptin ear containing, ARF binding protein 2; golgin A4; golgin A6 family, member C; golgin A8 family, member G; GRAM domain containing 4; GRB2-associated binding protein 2; growth arrest and DNA-damage-inducible, gamma; growth factor independent IB transcription repressor; growth factor receptor-bound protein 7; GS homeobox 1; GTPase activating protein (SH3 domain) binding protein 1 ; guanine nucleotide binding protein (G protein), alpha activating activity polypeptide O; guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 2; guanine nucleotide binding protein (G protein), beta 5; guanine nucleotide binding protein (G protein), beta polypeptide 1 ; guanylate binding protein 3; hairy/enhancer-of- split related with YRPW motif 1 ; HEAT repeat containing 5 A; heat shock protein 90kDa alpha (cytosolic), class A member 1 ; HECT domain containing 2; heme binding protein 1 ; heparan sulfate 6-O-sulfotransferase 1 ; hepatic leukemia factor; hepatitis A virus cellular receptor 2; heterochromatin protein 1, binding protein 3; hexamthylene bis-acetamide inducible 2;
hexosaminidase A (alpha polypeptide); high mobility group nucleosomal binding domain 3; high mobility group nucleosomal binding domain 4; histamine receptor H4; histone cluster 1, H3f; histone cluster 1, H4i; histone cluster 4, H4; homeobox A6; homeobox B8; homeobox containing 1; homeodomain interacting protein kinase 2; homer homolog 2 (Drosophila); homer homolog 3 (Drosophila); hyaluronan synthase 2; hyaluronan-mediated motility receptor (RHAMM);
hydroxyacid oxidase 2 (long chain); hydroxysteroid (17-beta) dehydrogenase 12; IMP (inosine 5 '-monophosphate) dehydrogenase 1 ; importin 5; ΓΝΟ80 homolog (S. cerevisiae); inositol 1,4,5- triphosphate receptor interacting protein; inositol 1,4,5 -triphosphate receptor, type 3; inositol hexakisphosphate kinase 2; insulin induced gene 2; integrin, alpha D; integrin, alpha E (antigen CD 103, human mucosal lymphocyte antigen 1 ; alpha polypeptide); integrin, beta 3 (platelet glycoprotein Ilia, antigen CD61); interferon induced transmembrane protein 1 (9-27); interferon regulatory factor 2; interferon regulatory factor 8; interleukin 12 receptor, beta 2; interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40);
interleukin 17C; interleukin 21 receptor; interleukin 22; intermediate filament tail domain containing 1 ; intracisternal A particle-promoted polypeptide; IQ motif and Sec7 domain 1 ; IQ motif and Sec7 domain 2; IQ motif and Sec7 domain 3; IQ motif containing H; jagged 1 (Alagille syndrome); kaptin (actin binding protein); kelch repeat and BTB (POZ) domain containing 13; kelch repeat and BTB (POZ) domain containing 6; kelch-like 12 (Drosophila); kelch-like 28 (Drosophila); kelch-like 36 (Drosophila); keratin 73; KIAA0020; KIAA0101; KIAA0125; KIAA0174; KIAA0240; KIAA0355; KIAA0415; KIAA0564; KIAA0776;
KIAA0892; KIAA1328; KIAA1549; KIAA1609; KIAA1671 ; killer cell lectin-like receptor subfamily K, member 1; kinesin family member 23; kinesin family member 26B; kinesin family member 3B; KIT ligand; Kruppel-like factor 13; Kruppel-like factor 14; Kv channel interacting protein 2; l(3)mbt-like 4 (Drosophila); lactase-like; Lbxcorl homolog (mouse); lectin, galactoside-binding, soluble, 13; leprecan-like 1; leucine rich repeat and Ig domain containing 1; leucine rich repeat and sterile alpha motif containing 1 ; leucine rich repeat containing 37, member A2; leucine rich repeat containing 37, member A4 (pseudogene); leucine rich repeat containing 42; leucine rich repeat containing 4C; leucine rich repeat containing 68; leucine rich repeat containing 8 family, member A; leucine-rich PPR-motif containing; leucine-rich repeats and calponin homology (CH) domain containing 3; leucyl/cystinyl aminopeptidase; ligand of numb-protein X 1; LIM domain and actin binding 1 ; LIM homeobox 5; limb bud and heart development homolog (mouse); lin-7 homolog C (C. elegans); lipase maturation factor 1; lipase, hepatic; lipocalin 6; Ion peptidase 2, peroxisomal; loss of heterozygosity, 12, chromosomal region 1; LSM11, U7 small nuclear RNA associated; LSM7 homolog, U6 small nuclear RNA associated (S. cerevisiae); LY6/PLAUR domain containing 5; lymphocyte antigen 86;
lymphocyte cytosolic protein 2 (SH2 domain containing leukocyte protein of 76kDa); lysine (K)- specific demethylase 3 A; lysozyme-like 1; macrophage scavenger receptor 1; mahogunin, ring finger 1; major histocompatibility complex, class I, G; major histocompatibility complex, class II, DM alpha; mannosidase, alpha, class 1A, member 1 ; mannosidase, beta A, lysosomal;
mannosyl (alpha-l,3-)-glycoprotein beta-l,2-N-acetylglucosaminyltransferase; mannosyl (beta- l,4-)-glycoprotein beta-l,4-N-acetylglucosaminyltransferase; mastermind-like 3 (Drosophila); matrilin 2; matrix metallopeptidase 16 (membrane-inserted); MAX dimerization protein 1 ; MAX gene associated; mediator complex subunit 13-like; mediator complex subunit 25; mediator complex subunit 27; mediator complex subunit 6; Mediterranean fever; melanoma cell adhesion molecule; membrane associated guanylate kinase, WW and PDZ domain containing 2; menage a trois homolog 1, cyclin H assembly factor (Xenopus laevis); mesencephalic astrocyte-derived neurotrophic factor; mesoderm specific transcript homolog (mouse); metadherin; metastasis associated 1 family, member 3; metaxin 3; methionine adenosyltransferase II, beta; methionine sulfoxide reductase B3; methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2-like; methyltransferase 11 domain containing 1; mevalonate (diphospho) decarboxylase; mex-3 homolog B (C. elegans); microsomal glutathione S-transferase 3; microtubule associated serine/threonine kinase 2; microtubule associated tumor suppressor candidate 2; microtubule- associated protein 1 light chain 3 beta 2; midnolin; mindbomb homolog 1 (Drosophila);
minichromosome maintenance complex component 8; mitochondrial ribosomal protein 63; mitochondrial ribosomal protein L41 ; mitochondrial ribosomal protein S I 1 ; mitochondrial ribosomal protein S25; mitochondrial translational release factor 1 ; mitogen-activated protein kinase 13; mitogen-activated protein kinase kinase 6; mitogen-activated protein kinase kinase kinase 14; MON2 homolog (S. cerevisiae); monoacylglycerol O-acyltransferase 2; MORN repeat containing 4; motilin; mucin 5B, oligomeric mucus/gel-forming; multimerin 2; multiple C2 domains, transmembrane 2; musashi homolog 2 (Drosophila); musculin; musculin; myelin- associated oligodendrocyte basic protein; myelodysplastic syndrome 2 translocation associated; myeloid cell leukemia sequence 1 (BCL2-related); myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); myocardin; myomesin (M-protein) 2, 165kDa; myomesin 1, 185kDa; myosin ID; myosin VIIA and Rab interacting protein; myosin VIIB; myosin XVA; myosin, heavy chain 13, skeletal muscle; myotubularin related protein 3; myotubularin related protein 9; myozenin 3; NACC family member 2, BEN and BTB (POZ) domain containing; N- acetylglucosamine- 1 -phosphodiester alpha-N-acetylglucosaminidase; N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase); N-acetyltransferase 8 (GCN5-related, putative); N-acylsphingosine amidohydrolase (acid ceramidase) 1; NADH dehydrogenase (ubiquinone) Fe- S protein 4, 18kDa (NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone) flavoprotein 3, lOkDa; NADPH oxidase 3; naked cuticle homolog 1 (Drosophila); NCK adaptor protein 2; NCK interacting protein with SH3 domain; N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3; NDRG family member 4; NECAP endocytosis associated 2; NEDD4 binding protein 2-like 1 ; neighbor of BRCA1 gene 2 (non-protein coding); neugrin, neurite outgrowth associated; neural cell adhesion molecule 2; neural precursor cell expressed, developmentally down-regulated 9; neuregulin 1 ; neuregulin 2; neurexophilin 1; neuroblastoma breakpoint family, member 1 1; neuroblastoma breakpoint family, member 15; neurocalcin delta;
neurocanthocytosis; neuron navigator 3; neuropeptide FF-amide peptide precursor; neuropilin (NRP) and tolloid (TLL)-like 1 ; neurotrophic tyrosine kinase, receptor, type 3; NFKB activating protein; NHS-like 1 ; nicotinamide nucleotide adenylyltransferase 3; NIMA (never in mitosis gene a)-related kinase 5; nitric oxide synthase 2, inducible; NK2 transcription factor related, locus 5 (Drosophila); NLR family, pyrin domain containing 5; NMDA receptor regulated 2; N- myc (and STAT) interactor; N-myristoyltransferase 2; non-SMC element 1 homolog (S.
cerevisiae); non-SMC element 2, MMS21 homolog (S. cerevisiae); NOP2/Sun domain family, member 6; nuclear factor I/A; nuclear mitotic apparatus protein 1 ; nuclear pore complex interacting protein-like 3 ; nuclear prelamin A recognition factor-like; nuclear protein localization 4 homolog (S. cerevisiae); nuclear receptor binding protein 2; nuclear receptor coactivator 2; nuclear receptor subfamily 1, group D, member 2; nuclear receptor subfamily 4, group A, member 2; nucleolin; nucleoporin 210kDa-like; nucleoporin 214kDa; NudC domain containing 1; nudix (nucleoside diphosphate linked moiety X)-type motif 11 ; numb homolog (Drosophila); odz, odd Oz/ten-m homolog 4 (Drosophila); olfactory receptor, family 4, subfamily F, member 29; olfactory receptor, family 4, subfamily F, member 4; olfactory receptor, family 4, subfamily F, member 5; olfactory receptor, family 5, subfamily AU, member 1 ; outer dense fiber of sperm tails 3-like 1 ; paired box 6; pantothenate kinase 2; papilin, proteoglycan-like sulfated
glycoprotein; PARK2 co-regulated; patched homolog 1 (Drosophila); PCF 11, cleavage and polyadenylation factor subunit, homolog (S. cerevisiae); PDS5, regulator of cohesion maintenance, homolog A (S. cerevisiae); PDZ and LIM domain 2 (mystique); pecanex homolog (Drosophila); peptidylprolyl isomerase A (cyclophilin A)-like 4B; perforin 1 (pore forming protein); pericentrin; periphilin 1 ; peroxisome proliferator-activated receptor alpha; peroxisome proliferator-activated receptor gamma, coactivator 1 beta; PHD finger protein 12; PHD finger protein 3; phenylalanyl-tRNA synthetase 2, mitochondrial; phosphatase and actin regulator 4; phosphatase and tensin homolog; phosphatidylethanolamine-binding protein 4;
phosphatidylinositol binding clathrin assembly protein; phosphatidylinositol glycan anchor biosynthesis, class L; phosphatidylinositol glycan anchor biosynthesis, class W;
phosphatidylinositol transfer protein, membrane-associated 2; phosphatidylinositol-3,4,5- trisphosphate-dependent Rac exchange factor 1; phosphatidylinositol-3,4,5-trisphosphate- dependent Rac exchange factor 2; phosphatidylinositol-4-phosphate 5-kinase, type I, beta;
phosphatidylinositol-4-phosphate 5-kinase, type I, gamma; phosphoglucomutase 5;
phosphoinositide-3-kinase, class 2, alpha polypeptide; phospholipase A2, group IVC (cytosolic, calcium-independent); phospholipase A2, group XIIB; phosphomannomutase 1; phosphorylase kinase, alpha 2 (liver); phosphorylase, glycogen, liver; phytanoyl-CoA dioxygenase domain containing 1 ; pituitary tumor-transforming 1 interacting protein; plasminogen-like B 1 ; platelet- activating factor acetylhydrolase lb, regulatory subunit 1 (45kDa); pleckstrin homology domain containing, family A member 5; pleckstrin homology domain containing, family G (with RhoGef domain) member 6; pleckstrin homology domain containing, family O member 1 ; pleomorphic adenoma gene-like 1 ; PMS1 postmeiotic segregation increased 1 (S. cerevisiae); polyamine modulated factor 1 binding protein 1 ; polybromo 1; polymerase (RNA) II (DNA directed) polypeptide J2; polymerase (RNA) II (DNA directed) polypeptide J3; polymerase (RNA) III (DNA directed) polypeptide B; polymerase (RNA) III (DNA directed) polypeptide F, 39 kDa; post-GPI attachment to proteins 2; potassium channel modulatory factor 1 ; potassium channel tetramerisation domain containing 13; potassium large conductance calcium-activated channel, subfamily M, alpha member 1 ; potassium large conductance calcium-activated channel, subfamily M, beta member 4; POU class 2 associating factor 1; POU class 2 homeobox 2; PR domain containing 11 ; PR domain containing 4; pre-B-cell leukemia homeobox 4; pregnancy- zone protein; presenilin enhancer 2 homolog (C. elegans); proenkephalin; progesterone receptor membrane component 2; progestin and adipoQ receptor family member V; progestin and adipoQ receptor family member VI; prolactin; proline rich 7 (synaptic); proline rich Gla (G- carboxyglutamic acid) 4 (transmembrane); proline-rich protein BstNI subfamily 2; prolyl 4- hydroxylase, alpha polypeptide I; prolyl 4-hydroxylase, alpha polypeptide III; prostaglandin F receptor (FP); prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase); protease, serine, 1 (trypsin 1); protease, serine, 12 (neurotrypsin, motopsin); proteasomal ATPase-associated factor 1; proteasome (prosome, macropain) subunit, alpha type, 5; proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional peptidase 2); protein kinase C, beta; protein kinase C, eta; protein kinase Dl ; protein kinase, AMP-activated, gamma 2 non-catalytic subunit; protein phosphatase 1, regulatory (inhibitor) subunit 16A;
protein phosphatase 1, regulatory (inhibitor) subunit 3B; protein phosphatase 2, regulatory subunit B, alpha; protein phosphatase 2, regulatory subunit B", gamma; protein phosphatase 3, catalytic subunit, gamma isozyme; protein phosphatase 5, catalytic subunit; protein phosphatase, Mg2+/Mn2+ dependent, 1H; protein tyrosine phosphatase, non-receptor type 23; protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 1 ; protein tyrosine phosphatase, receptor type, H; protocadherin alpha 2; protocadherin beta 1;
protocadherin beta 12; protocadherin beta 13; protocadherin beta 18 pseudogene; protocadherin gamma subfamily A, 12; protocadherin gamma subfamily A, 12; PTPRF interacting protein, binding protein 1 (liprin beta 1); pumilio homolog 2 (Drosophila); purinergic receptor P2X, ligand-gated ion channel, 7; pyrophosphatase (inorganic) 2; quaking homolog, KH domain RNA binding (mouse); quiescin Q6 sulfhydryl oxidase 1 ; quinoid dihydropteridine reductase; Rab interacting lysosomal protein-like 1 ; RAB, member of RAS oncogene family-like 2A; RAB IB, member RAS oncogene family; RAB23, member RAS oncogene family; RAB27A, member RAS oncogene family; RAB27B, member RAS oncogene family; RAB28, member RAS oncogene family; RAB30, member RAS oncogene family; RAB40B, member RAS oncogene family; RAB4B, member RAS oncogene family; RAB8A, member RAS oncogene family; RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae); RAD52 homolog (S. cerevisiae); Ral GTPase activating protein, alpha subunit 1 (catalytic); Rap guanine nucleotide exchange factor (GEF) 3; RAPIB, member of RAS oncogene family; Ras association (RalGDS/AF-6) domain family ( -terminal) member 7; ras homolog gene family, member Q; RAS protein activator like 2; receptor accessory protein 3; regenerating islet-derived family, member 4; regulator of chromosome condensation 1; regulator of chromosome condensation 2; regulator of G-protein signaling 20; regulator of G-protein signaling 7; regulator of G-protein signaling 8; regulatory associated protein of MTOR, complex 1 ; reprimo-like; resistance to inhibitors of cholinesterase 3 homolog (C. elegans); retinoblastoma binding protein 6; retinoic acid receptor, alpha; REV3- like, catalytic subunit of DNA polymerase zeta (yeast); REX1, RNA exonuclease 1 homolog (S. cerevisiae); REX1, RNA exonuclease 1 homolog (S. cerevisiae)-like 2 (pseudogene); Rho GTPase activating protein 17; Rho GTPase activating protein 17; Rho GTPase activating protein 22; Rho GTPase activating protein 28; rhomboid 5 homolog 2 (Drosophila); Rho-related BTB domain containing 2; ribonuclease HI ; ribonuclease P/MRP 40kDa subunit; ribonucleoprotein, PTB-binding 2; ribonucleotide reductase M2 B (TP53 inducible); ribosomal modification protein rimK-like family member B; ribosomal protein L14; ribosomal protein L31 ; ribosomal protein L36a-like; ribosomal protein L7; ribosomal protein SA pseudogene 58; ribosomal protein, large, PI ; ring finger protein 135; ring finger protein 144B; ring finger protein 165; ring finger protein 183; ring finger protein 208; ring finger protein 213; ring finger protein 220; ring finger protein, transmembrane 1; RTNGl and YY1 binding protein; RNA binding motif protein 26; ROD1 regulator of differentiation 1 (S. pombe); RPGRIP 1 -like; RUN and FYVE domain containing 3; RUN domain containing 2C; runt-related transcription factor 2; SI 00 calcium binding protein Al l; SI 00 calcium binding protein A4; SAFB-like, transcription modulator; sal-like 1
(Drosophila); Salvador homolog 1 (Drosophila); sarcalumenin; sarcosine dehydrogenase; SCAN domain containing 3; scavenger receptor class B, member 1 ; scinderin; secretagogin, EF-hand calcium binding protein; secreted protein, acidic, cysteine-rich (osteonectin); septin 9; serine racemase; serine/threonine kinase 17a; serine/threonine kinase 17b; serine/threonine kinase 24 (STE20 homolog, yeast); serine/threonine kinase 4; serine/threonine/tyrosine interacting-like 1 ; serpin peptidase inhibitor, clade B (ovalbumin), member 1 ; SET and MYND domain containing 3; SET binding protein 1 ; SET domain containing (lysine methyltransferase) 7; SET domain containing 4; SFT2 domain containing 1 ; SGT1, suppressor of G2 allele of SKP1 (S. cerevisiae); SH3 and PX domains 2 A; SH3 domain containing 19; SHC (Src homology 2 domain containing) family, member 4; short chain dehydrogenase/reductase family 39U, member 1; sialic acid binding Ig-like lectin 5; sideroflexin 5; signal peptidase complex subunit 2 homolog (S. cerevisiae); signal recognition particle 14kDa (homologous Alu RNA binding protein); SIK family kinase 3; sine oculis binding protein homolog (Drosophila); SLAM family member 7; SMAD family member 6; small G protein signaling modulator 3; small nuclear
ribonucleoprotein polypeptide A'; small VCP/p97-interacting protein; SMEK homolog 1, suppressor of mekl (Dictyostelium); smoothelin-like 2; sodium channel, voltage gated, type VIII, alpha subunit; sodium channel, voltage-gated, type IV, alpha subunit; solute carrier family 10 (sodium/bile acid cotransporter family), member 5; solute carrier family 12
(sodium/potassium/chloride transporters), member 1; solute carrier family 16, member 1 (monocarboxylic acid transporter 1); solute carrier family 16, member 3 (monocarboxylic acid transporter 4); solute carrier family 16, member 4 (monocarboxylic acid transporter 5); solute carrier family 17, member 9; solute carrier family 2 (facilitated glucose transporter), member 11 ; solute carrier family 2 (facilitated glucose transporter), member 9; solute carrier family 22, member 23; solute carrier family 22, member 23; solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3; solute carrier family 25, member 13 (citrin); solute carrier family 25, member 30; solute carrier family 26 (sulfate transporter), member 2; solute carrier family 27 (fatty acid transporter), member 5; solute carrier family 35, member B3; solute carrier family 35, member F5; solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; solute carrier family 39 (zinc transporter), member 14; solute carrier family 45, member 3; solute carrier family 46, member 3; solute carrier organic anion transporter family, member 3A1 ; solute carrier organic anion transporter family, member 5A1; somatostatin receptor 4; sorting nexin 29; sorting nexin family member 30; spastic paraplegia 7 (pure and complicated autosomal recessive); speedy homolog El (Xenopus laevis); spermatogenesis associated 13; spermatogenesis associated 8; spindlin family, member 2A; spinster homolog 3 (Drosophila); spleen focus forming virus (SFFV) proviral integration oncogene spil; spleen tyrosine kinase; splicing factor, arginine/serine-rich 1 ; splicing factor, arginine/serine-rich 12; spondin 2, extracellular matrix protein; sprouty homolog 1, antagonist of FGF signaling (Drosophila); SRY (sex determining region Y)-box 1 ; SRY (sex determining region Y)-box 12; SRY (sex determining region Y)-box 21 ; SRY (sex determining region Y)-box 4; SRY (sex determining region Y)-box 7; SRY (sex determining region Y)-box 9; ST3 beta-galactoside alpha-2,3-sialyltransferase 5; staufen, RNA binding protein, homolog 1 (Drosophila); staufen, RNA binding protein, homolog 2
(Drosophila); STE20-related kinase adaptor alpha; sterile alpha motif domain containing 4A; sterol O-acyltransferase 2; sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, S.
cerevisiae)-like; stress-associated endoplasmic reticulum protein family member 2; stromal antigen 3 -like 2; stromal interaction molecule 1 ; sulfatase 2; sulfotransferase family, cytosolic, 1A, phenol-preferring, member 3; SUMO l/sentrin specific peptidase 6; SUM01/sentrin/SMT3 specific peptidase 2; supervillin; suppressor of cytokine signaling 6; survival of motor neuron 2, centromeric; sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 ; SWAP switching B-cell complex 70kDa subunit; SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily e, member 1 ; synapsin III; synaptic Ras GTPase activating protein 1 homolog (rat); synaptonemal complex protein 2-like; synaptosomal- associated protein, 47kDa; synaptotagmin II; synaptotagmin IV; synaptotagmin IX;
synaptotagmin VII; synaptotagmin X; syntaxin 4; syntaxin binding protein 5 (tomosyn);
syntrophin, beta 1 (dystrophin-associated protein Al, 59kDa, basic component 1); T cell receptor alpha constant; T cell receptor gamma variable 10 (non-functional); T cell receptor gamma variable 8; TAF15 R A polymerase II, TATA box binding protein (TBP)-associated factor, 68kDa; TAO kinase 3; TBC1 domain family, member 21 ; TBC1 domain family, member 28; TBC1 domain family, member 4; TBC1 domain family, member 5; TBC1 domain family, member 8 (with GRAM domain); T-box 3; T-cell leukemia/lymphoma 1A; t-complex 1 1 homolog (mouse); TEA domain family member 1 (SV40 transcriptional enhancer factor);
tectonin beta-propeller repeat containing 2; testis expressed 10; tetraspanin 1 1; tetraspanin 4; tetraspanin 9; tetratricopeptide repeat domain 23-like; tetratricopeptide repeat domain 39B; tetratricopeptide repeat domain 4; tetratricopeptide repeat domain 7B; tetratricopeptide repeat domain 8; TGFB-induced factor homeobox 2; THO complex 2; thrombospondin 2; thyrotropin- releasing hormone degrading enzyme; tight junction protein 2 (zona occludens 2); TNF receptor- associated factor 5; toll-like receptor 1 ; topoisomerase (DNA) I, mitochondrial; torsin A interacting protein 2; tousled-like kinase 1 ; tousled-like kinase 2; TOX high mobility group box family member 3; TRAF3 interacting protein 2; trafficking protein particle complex 9;
transcription elongation factor A (SII), 1 ; transcription elongation factor A (Sll)-like 1 ;
transcription factor 12; transcription factor 4; transcription factor 7-like 1 (T-cell specific, HMG- box); transcription factor Dp-1 ; transducin (beta)-like 1 X-linked receptor 1; transducin-like enhancer of split 3 (E(spl) homolog, Drosophila); translocase of inner mitochondrial membrane 23 homolog B (yeast); translocation associated membrane protein 1 ; transmembrane 6 superfamily member 2; transmembrane and coiled-coil domain family 1 ; transmembrane and coiled-coil domains 4; transmembrane and coiled-coil domains 5A; transmembrane anterior posterior transformation 1; transmembrane protease, serine 9; transmembrane protein 121 ;
transmembrane protein 121 ; transmembrane protein 132B; transmembrane protein 132D;
transmembrane protein 14C; transmembrane protein 154; transmembrane protein 164;
transmembrane protein 196; transmembrane protein 2; transmembrane protein 30B;
transmembrane protein 59; transmembrane protein 64; transmembrane protein 90B; transportin 3; tribbles homolog 1 (Drosophila); trichorhinophalangeal syndrome I; trinucleotide repeat containing 18; TRIO and F-actin binding protein; tripartite motif-containing 16; tripartite motif- containing 36; tripartite motif-containing 4; tRNA methyltransferase 12 homolog (S. cerevisiae); tRNA splicing endonuclease 2 homolog (S. cerevisiae); tropomodulin 3 (ubiquitous); troponin I type 3 (cardiac); TSPY-like 2; tubby homolog (mouse); tuberous sclerosis 1; tubulin, alpha lc; tubulin, alpha lc; tubulin, beta 2A; tubulin, beta 2B; tumor necrosis factor (ligand) superfamily, member 4; tumor necrosis factor receptor superfamily, member 10a; tumor necrosis factor receptor superfamily, member 10b; tumor necrosis factor, alpha-induced protein 2; tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; tyrosine kinase, non-receptor, 2; tyrosylprotein sulfotransferase 1; UBA domain containing 1 ; UBA domain containing 2; ubiquitin fusion degradation 1 like (yeast); ubiquitin protein ligase E3 component n-recognin 3 (putative); ubiquitin specific peptidase 2; ubiquitin specific peptidase 24; ubiquitin specific peptidase 3; ubiquitin-conjugating enzyme E2I (UBC9 homolog, yeast); ubiquitin-like 7 (bone marrow stromal cell-derived); U-box domain containing 5; UDP- GlcNAc:betaGal beta-l,3-N-acetylglucosaminyltransferase 7; UDP-glucose ceramide glucosyltransferase; UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 10 (GalNAc-TIO); UDP-N-acetyl-alpha-D- galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7); UEV and lactate/malate dehyrogenase domains; unc-13 homolog C (C. elegans); unc-93 homolog B l (C. elegans); unkempt homolog (Drosophila); Vac 14 homolog (S. cerevisiae); vaccinia related kinase 2; vaccinia related kinase 3; vacuolar protein sorting 13 homolog A (S. cerevisiae);
vacuolar protein sorting 18 homolog (S. cerevisiae); vascular endothelial growth factor A;
vascular endothelial zinc finger 1 ; vasohibin 1 ; vestigial like 3 (Drosophila); vestigial like 4 (Drosophila); v-ets erythroblastosis virus E26 oncogene homolog 1 (avian); vinculin; vitamin K epoxide reductase complex, subunit 1 -like 1 ; vitelline membrane outer layer 1 homolog
(chicken); v-maf musculoaponeurotic fibrosarcoma oncogene homolog G (avian); v-myc myelocytomatosis viral oncogene homolog (avian); von Hippel-Lindau tumor suppressor; von Willebrand factor; v-ral simian leukemia viral oncogene homolog A (ras related); v-rel reticuloendotheliosis viral oncogene homolog (avian); WAS protein family homolog 2 pseudogene; WAS protein homolog associated with actin, golgi membranes and microtubules; WD repeat domain 44; WD repeat domain 70; Werner helicase interacting protein 1 ; wingless- type MMTV integration site family, member 1 ; wingless-type MMTV integration site family, member 9A; WW domain binding protein 4 (formin binding protein 21); WWC family member 3; XK, Kell blood group complex subunit-related family, member 5; XK, Kell blood group complex subunit-related family, member 6; X-prolyl aminopeptidase (aminopeptidase P) 3, putative; yippee-like 3 (Drosophila); zeta-chain (TCR) associated protein kinase 70kDa; zinc and ring finger 1 ; zinc and ring finger 2; zinc and ring finger 4; zinc finger and AT hook domain containing; zinc finger and BTB domain containing 25; zinc finger and BTB domain containing 26; zinc finger and BTB domain containing 48; zinc finger and SCAN domain containing 10; zinc finger and SCAN domain containing 18; zinc finger CCCH-type containing 7A; zinc finger E-box binding homeobox 1; zinc finger protein 117; zinc finger protein 136; zinc finger protein 155; zinc finger protein 165; zinc finger protein 17; zinc finger protein 184; zinc finger protein 2 homolog (mouse); zinc finger protein 205; zinc finger protein 207; zinc finger protein 23 (KOX 16); zinc finger protein 26; zinc finger protein 28; zinc finger protein 28 homolog (mouse); zinc finger protein 280D; zinc finger protein 354A; zinc finger protein 383; zinc finger protein 404; zinc finger protein 418; zinc finger protein 423; zinc finger protein 426; zinc finger protein 429; zinc finger protein 432; zinc finger protein 461; zinc finger protein 493; zinc finger protein 507; zinc finger protein 534; zinc finger protein 544; zinc finger protein 56; zinc finger protein 560; zinc finger protein 561 ; zinc finger protein 566; zinc finger protein 582; zinc finger protein 584; zinc finger protein 587; zinc finger protein 594; zinc finger protein 596; zinc finger protein 599; zinc finger protein 600; zinc finger protein 613; zinc finger protein 623; zinc finger protein 643; zinc finger protein 655; zinc finger protein 66; zinc finger protein 667; zinc finger protein 681 ; zinc finger protein 689; zinc finger protein 704; zinc finger protein 71 ; zinc finger protein 710; zinc finger protein 726; zinc finger protein 726; zinc finger protein 747; zinc finger protein 768; zinc finger protein 770; zinc finger protein 791; zinc finger protein 8; zinc finger protein 83; zinc finger protein 861 (pseudogene); zinc finger protein 880; zinc finger protein 90; zinc finger protein 91 ; zinc finger protein 99; zinc finger protein, multitype 2; zinc finger, BED-type containing 4; zinc finger, BED-type containing 5; zinc finger, CCHC domain containing 2; zinc finger, CCHC domain containing 3; zinc finger, DHHC-type containing 2; zinc finger, HIT type 6; zinc finger, imprinted 2; zinc finger, matrin type 2; zinc finger, MYND-type containing 8; and zinc finger, SWIM-type containing 5.
[0030] In one embodiment, IncRNA transcripts or genes found at or near a chromosomal locus associated with malignancy are targeted for the treatment, diagnosis or therapeutic outcomes. For example, IncRNA transcripts found downstream of the coding gene, septin 9, may be targeted in the treatment or diagnosis of cancers such as ovarian cancer.
[0031] In one embodiment, IncRNA transcripts are encoded between two coding genes known to have an association with malignancy or disease and these are preferred targets of the invention. For example, several IncRNA genes are found coded between v-myc genes and gasdermin C, a coding gene whose product is associated with metastatic melanoma. [0032] In one embodiment, IncRNA transcripts or genes are encoded either upstream or downstream (nearest neighbor) to one or more pseudogenes. These IncRNA are also preferred targets for the therapeutic outcomes described herein.
[0033] Protein coding genes found downstream of IncRNA genes or transcripts may be targeted by any of the methods disclosed herein. These genes include, but are not limited to 1- acylglycerol-3 -phosphate O-acyltransferase 5 (lysophosphatidic acid acyltransferase, epsilon); 1- aminocyclopropane-l-carboxylate synthase homolog (Arabidopsis)(non-functional)-like; 3- hydroxyisobutyrate dehydrogenase; 3-phosphoinositide dependent protein kinase- 1 ; A kinase (PRKA) anchor protein 11 ; A kinase (PRKA) anchor protein 8-like; abhydrolase domain containing 6; acetoacetyl-CoA synthetase; acid phosphatase 5, tartrate resistant; acidic (leucine- rich) nuclear phosphoprotein 32 family, member E; aconitase 2, mitochondrial; actin related protein 2/3 complex, subunit 2, 34kDa; activating transcription factor 7; activating transcription factor 7 interacting protein 2; activator of basal transcription 1; acyl-CoA oxidase 1, palmitoyl; acyl-CoA synthetase long-chain family member 3; acyl-CoA synthetase medium-chain family member 1; acyl-CoA synthetase short-chain family member 1 ; acyl-CoA thioesterase 13; acyl- malonyl condensing enzyme 1-like 2; acylphosphatase 2, muscle type; ADAM metallopeptidase with thrombospondin type 1 motif, 14; adaptor-related protein complex 2, beta 1 subunit;
adaptor-related protein complex 3, beta 2 subunit; adaptor-related protein complex 4, epsilon 1 subunit; additional sex combs like 1 (Drosophila); ADP-ribosylation factor-like 17A; ADP- ribosylation factor-like 5B; adrenergic, alpha- 1B-, receptor; AF4/FMR2 family, member 1; aldehyde dehydrogenase 1 family, member A3 ; aldo-keto reductase family 1 , member C 1 (dihydrodiol dehydrogenase 1 ; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase); alkB, alkylation repair homolog 5 (E. coli); alpha 1,4-galactosyltransferase; alpha-2-macroglobulin; alpha-2-macroglobulin-like 1; amino-terminal enhancer of split; amphiphysin; amyloid beta (A4) precursor protein; amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein; angel homolog 1 (Drosophila); ankyrin 3, node of Ranvier (ankyrin G); ankyrin repeat and sterile alpha motif domain containing IB; ankyrin repeat domain 1 1; ankyrin repeat domain 20 family, member A3; ankyrin repeat domain 42; ankyrin repeat domain 50; annexin A4;
apoptosis enhancing nuclease; apoptosis inhibitor 5; aquaporin 9; aquarius homolog (mouse); arachidonate 15 -lipoxygenase; ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 1 ; ArfGAP with SH3 domain, ankyrin repeat and PH domain 1; arginine vasopressin receptor 1A; argininosuccinate synthase 1; ariadne homolog 2 (Drosophila); ariadne homolog, ubiquitin- conjugating enzyme E2 binding protein, 1 (Drosophila); ARP3 actin-related protein 3 homolog C (yeast); arrestin domain containing 1 ; arrestin domain containing 4; aryl-hydrocarbon receptor nuclear translocator 2; asparagine-linked glycosylation 10, alpha- 1,2-glucosyltransferase homolog B (yeast); asparagine-linked glycosylation 12, alpha- 1,6-mannosyltransferase homolog (S. cerevisiae); asparaginyl-tRNA synthetase 2, mitochondrial (putative); AT rich interactive domain 1A (SWI-like); AT rich interactive domain IB (SWIl-like); AT rich interactive domain 3B (BRIGHT-like); ataxin 2-like; ATG12 autophagy related 12 homolog (S. cerevisiae); ATP binding domain 4; ATPase, class VI, type 1 1A; ATP-binding cassette, sub-family B
(MDR/TAP), member 1 ; ATP-binding cassette, sub-family C (CFTR/MRP), member 4; ATX1 antioxidant protein 1 homolog (yeast); AU R A binding protein/enoyl-CoA hydratase; B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB; BAH domain and coiled-coil containing 1 ; BAI1 -associated protein 2-like 1 ; basic helix-loop-helix family, member e23; basonuclin 1 ; bassoon (presynaptic cytomatrix protein); BCDIN3 domain containing; B-cell translocation gene 4; Bcl2 modifying factor; BCL6 corepressor; beta-l,4-N- acetyl-galactosaminyl transferase 4; Bloom syndrome, RecQ helicase-like; bobby sox homolog (Drosophila); bol, boule-like (Drosophila); bradykinin receptor B2; brain and acute leukemia, cytoplasmic; brain and reproductive organ-expressed (TNFRSF1A modulator); brain protein 44- like; brain-derived neurotrophic factor; breast cancer metastasis-suppressor 1-like; bromodomain and PHD finger containing, 3; bromodomain PHD finger transcription factor; BUD 13 homolog (S. cerevisiae); butyrophilin, subfamily 1, member Al ; Clq and tumor necrosis factor related protein 5; cadherin 12, type 2 (N-cadherin 2); cadherin 6, type 2, K-cadherin (fetal kidney); cadherin 9, type 2 (Tl -cadherin); calcineurin binding protein 1; calcineurin-like phosphoesterase domain containing 1; caldesmon 1 ; calmin (calponin-like, transmembrane); calpain 13; calponin 1, basic, smooth muscle; calsyntenin 2; cAMP responsive element binding protein 5; CAP, adenylate cyclase-associated protein, 2 (yeast); carbohydrate (chondroitin 4) sulfotransferase 11 ; carbonyl reductase 3; carboxypeptidase Al (pancreatic); carboxypeptidase A5; carboxypeptidase A6; cardiolipin synthase 1 ; carnosine dipeptidase 1 (metallopeptidase M20 family); casein kinase 1, delta; CASP2 and RIPKl domain containing adaptor with death domain; caspase 3, apoptosis- related cysteine peptidase; catalase; catenin (cadherin-associated protein), alpha 3; catenin (cadherin-associated protein), beta 1, 88kDa; cathepsin B; CD19 molecule; CD3e molecule, epsilon (CD3-TCR complex); CD44 molecule (Indian blood group); CD5 molecule; CD7 molecule; CD9 molecule; CD93 molecule; CD97 molecule; CDC42 effector protein (Rho GTPase binding) 4; CDK5 regulatory subunit associated protein 2; cell division cycle 27 homolog (S. cerevisiae); cell division cycle and apoptosis regulator 1 ; cellular retinoic acid binding protein 2; centromere protein V; ceramide kinase; cerebellin 2 precursor; chemokine (C- C motif) ligand 15; chemokine (C-X-C motif) receptor 5; chloride intracellular channel 5;
CHMP family, member 7; chondrolectin; chromatin modifying protein 2B; chromobox homolog 7; chromodomain helicase DNA binding protein 3; chromodomain helicase DNA binding protein 4; chromodomain helicase DNA binding protein 6; chromodomain protein, Y-like 2;
chromosome 1 open reading frame 222; chromosome 1 open reading frame 43; chromosome 1 open reading frame 97; chromosome 10 open reading frame 11 ; chromosome 10 open reading frame 140; chromosome 1 1 open reading frame 2; chromosome 1 1 open reading frame 61 ; chromosome 1 1 open reading frame 73; chromosome 12 open reading frame 49; chromosome 12 open reading frame 70; chromosome 13 open reading frame 28; chromosome 14 open reading frame 104; chromosome 14 open reading frame 126; chromosome 14 open reading frame 145; chromosome 14 open reading frame 153; chromosome 14 open reading frame 159; chromosome 14 open reading frame 177; chromosome 14 open reading frame 182; chromosome 14 open reading frame 183; chromosome 15 open reading frame 41 ; chromosome 15 open reading frame 54; chromosome 15 open reading frame 57; chromosome 16 open reading frame 89;
chromosome 17 open reading frame 108; chromosome 17 open reading frame 57; chromosome 17 open reading frame 72; chromosome 17 open reading frame 82; chromosome 19 open reading frame 23; chromosome 19 open reading frame 42; chromosome 2 open reading frame 40;
chromosome 20 open reading frame 1 17; chromosome 20 open reading frame 24; chromosome 21 open reading frame 57; chromosome 3 open reading frame 1; chromosome 3 open reading frame 35; chromosome 4 open reading frame 19; chromosome 4 open reading frame 34;
chromosome 5 open reading frame 25; chromosome 5 open reading frame 52; chromosome 5 open reading frame 58; chromosome 6 open reading frame 145; chromosome 6 open reading frame 170; chromosome 6 open reading frame 195; chromosome 6 open reading frame 223; chromosome 7 open reading frame 33; chromosome 7 open reading frame 44; chromosome 7 open reading frame 50; chromosome 8 open reading frame 34; chromosome 8 open reading frame 38; chromosome 9 open reading frame 153; chromosome 9 open reading frame 3;
chromosome 9 open reading frame 30; chromosome 9 open reading frame 93; chronic lymphocytic leukemia up-regulated 1 opposite strand; chymase 1, mast cell; cingulin-like 1 ; clathrin interactor 1; claudin 16; coactosin-like 1 (Dictyostelium); coatomer protein complex, subunit epsilon; COBW domain containing 3; coenzyme Q9 homolog (S. cerevisiae); coiled-coil domain containing 102B; coiled-coil domain containing 147; coiled-coil domain containing 155; coiled-coil domain containing 28A; coiled-coil domain containing 8; collagen, type XXIII, alpha 1; COMM domain containing 10; COMM domain containing 6; core-binding factor, runt domain, alpha subunit 2; translocated to, 3; cornichon homolog 2 (Drosophila); cortactin; C- terminal binding protein 1 ; C-type lectin domain family 18, member C; CUB and Sushi multiple domains 1; cullin 7; cyclin B2; cyclin Y-like 2; cyclin-dependent kinase 6; cystatin F
(leukocy statin); cysteine sulfinic acid decarboxylase; cysteine-rich hydrophobic domain 2; cysteinyl-tRNA synthetase; cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-N-acetylneuraminate monooxygenase) pseudogene; cytochrome P450, family 4, subfamily F, polypeptide 2; cytokine receptor-like factor 3; cytoplasmic FMR1 interacting protein 2; DEAD (Asp-Glu-Ala-As) box polypeptide 19A; DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11 (CHLl-like helicase homolog, S. cerevisiae); DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 1 1 like 8; DEAH (Asp-Glu-Ala-His) box polypeptide 9; death effector domain containing 2; dedicator of cytokinesis 1; de-etiolated homolog 1 (Arabidopsis); defensin, beta 1; deiodinase, iodothyronine, type III; delta-like 4 (Drosophila); DE /MADD domain containing 4A; DENN/MADD domain containing 4B; density-regulated protein; deoxyuridine
triphosphatase; DEP domain containing 6; diacylglycerol kinase, eta; diacylglycerol lipase, alpha; diacylglycerol O-acyltransferase homolog 2 (mouse); diaphanous homolog 1
(Drosophila); diaphanous homolog 1 (Drosophila); DIP2 disco-interacting protein 2 homolog A (Drosophila); DIS3 mitotic control homolog (S. cerevisiae)-like; discs, large (Drosophila) homolog-associated protein 1; discs, large homolog 1 (Drosophila); discs, large homolog 5 (Drosophila); DnaJ (Hsp40) homolog, subfamily B, member 1; DnaJ (Hsp40) homolog, subfamily C, member 3; DnaJ (Hsp40) homolog, subfamily C, member 7; dopa decarboxylase (aromatic L-amino acid decarboxylase); dopey family member 1 ; dorsal root ganglia homeobox; DOTl-like, histone H3 methyltransferase (S. cerevisiae); doublecortin-like kinase 1 ; doublesex and mab-3 related transcription factor 2; dpy-19-like 1 (C. elegans); dpy-19-like 3 (C. elegans); drebrin-like; dual specificity phosphatase 16; dual specificity phosphatase 6; dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2; dynein, cytoplasmic 1, light intermediate chain 2; dynein, cytoplasmic, light polypeptide pseudogene; dynein, light chain, LC8-type 1; dynein, light chain, LC8-type 2; dysbindin (dystrobrevin binding protein 1) domain containing 1 ; E2F transcription factor 2; early growth response 4; ecotropic viral integration site 5-like; EGF, latrophilin and seven transmembrane domain containing 1 ; egf-like module containing, mucin- like, hormone receptor-like 2; egl nine homolog 1 (C. elegans); egl nine homolog 2 (C. elegans); ELKS/RAB6-interacting/CAST family member 1; ELKS/RAB6-interacting/CAST family member 1 ; elongation factor Tu GTP binding domain containing 1 ; endoplasmic reticulum-golgi intermediate compartment (ERGIC) 1; endosulfine alpha; engrailed homeobox 1 ; enolase superfamily member 1; epiregulin; epithelial cell transforming sequence 2 oncogene; epithelial membrane protein 1 ; ets variant 6; eukaryotic translation initiation factor IB; eukaryotic translation initiation factor 2 alpha kinase 4; eukaryotic translation initiation factor 3, subunit H; eukaryotic translation initiation factor 5; exocyst complex component 4; exocyst complex component 8; exostoses (multiple)-like 3; extracellular leucine-rich repeat and fibronectin type III domain containing 2; eyes shut homolog (Drosophila); family with sequence similarity 100, member A; family with sequence similarity 103, member Al; family with sequence similarity 105, member B; family with sequence similarity 108, member B l; family with sequence similarity 1 11, member A; family with sequence similarity 1 13, member B; family with sequence similarity 125, member A; family with sequence similarity 125, member B; family with sequence similarity 138, member D; family with sequence similarity 156, member A;
family with sequence similarity 158, member A; family with sequence similarity 171, member Al; family with sequence similarity 178, member B; family with sequence similarity 179, member B; family with sequence similarity 18, member B2; family with sequence similarity 181, member B; family with sequence similarity 186, member B; family with sequence similarity 21, member A; family with sequence similarity 27-like; family with sequence similarity 35, member A; family with sequence similarity 59, member A; family with sequence similarity 63, member B; family with sequence similarity 84, member B; family with sequence similarity 86, member B2; family with sequence similarity 92, member Al ; farnesyltransferase, CAAX box, beta; fatty acid amide hydrolase 2; fatty acid binding protein 6, ileal; fatty acyl CoA reductase 2; F-box and leucine-rich repeat protein 14; F-box and leucine-rich repeat protein 22; F-box and leucine-rich repeat protein 22; F-box and WD repeat domain containing 8; F-box protein 15; F-box protein 25; fer (fps/fes related) tyrosine kinase; FERM domain containing 4A; FERM, RhoGEF
(ARHGEF) and pleckstrin domain protein 1 (chondrocyte-derived); FH2 domain containing 1 ; fibronectin type III domain containing 3B; FIC domain containing; FK506 binding protein IB, 12.6 kDa; FK506 binding protein 4, 59kDa; forkhead box CI; forkhead box F l; forkhead box N3; formin-like 1 ; formyl peptide receptor 3; Friend leukemia virus integration 1; frizzled homolog 10 (Drosophila); frizzled homolog 6 (Drosophila); fructosamine 3 kinase related protein; fucosyltransferase 10 (alpha (1,3) fucosyltransferase); fucosyltransferase 5 (alpha (1,3) fucosyltransferase); FXYD domain containing ion transport regulator 2; FY oncogene related to SRC, FGR, YES; G kinase anchoring protein 1 ; G protein-coupled estrogen receptor 1; G protein-coupled receptor 107; G protein-coupled receptor 132; G protein-coupled receptor 63; G2/M-phase specific E3 ubiquitin ligase; GABA(A) receptors associated protein like 3
(pseudogene); galanin receptor 2; gametogenetin binding protein 2; ganglioside induced differentiation associated protein 2; gap junction protein, gamma 3, 30.2kDa; gasdermin C; gastric inhibitory polypeptide receptor; GATA binding protein 6; GATA zinc finger domain containing 2A; gelsolin; general transcription factor IIH, polypeptide 2, 44kDa; general transcription factor IIIC, polypeptide 1 , alpha 220kDa; general transcription factor IIIC, polypeptide 5, 63kDa; glioblastoma amplified sequence; glucan (1,4-alpha-), branching enzyme 1; glucocorticoid deficiency 3; glutamate receptor, ionotropic, delta 1 ; glutamate receptor, ionotropic, kainate 4; glutamate receptor, ionotropic, kainate 5; glutamate receptor, metabotropic 4; glutamate-rich 1; glutamic-oxaloacetic transaminase 1-like 1; glutaminase; glutamine and serine rich 1 ; glutaredoxin 3; glutaredoxin 5; glycerol-3 -phosphate dehydrogenase 2
(mitochondrial); glycerophosphodiester phosphodiesterase domain containing 3; glycine C- acetyltransferase; glycine cleavage system protein H (aminomethyl carrier); glycophorin E (MNS blood group); golgi-associated, gamma adaptin ear containing, ARF binding protein 2; golgin A6 family, member A; golgin A6 family, member D; G-protein signaling modulator 1 (AGS3-like, C. elegans); GRAM domain containing 1C; GRAM domain containing 4; growth differentiation factor 6; growth hormone receptor; GTPase activating protein (SH3 domain) binding protein 1 ; guanidinoacetate N-methyltransferase; guanine nucleotide binding protein (G protein), alpha activating activity polypeptide O; guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 1 ; guanine nucleotide binding protein (G protein), beta polypeptide 1 ; guanine nucleotide binding protein (G protein), gamma 2; guanine nucleotide binding protein (G protein), gamma 7; guanylate binding protein 1, interferon- inducible, 67kDa; HEAT repeat containing 3; HEAT repeat containing 4; heat shock 27kDa protein 2;
hematological and neurological expressed 1 -like; hematopoietic SH2 domain containing; heme binding protein 1 ; heparan sulfate (glucosamine) 3-O-sulfotransferase 3B1 ; hepatocyte nuclear factor 4, gamma; heterochromatin protein 1 , binding protein 3 ; heterogeneous nuclear ribonucleoprotein C (C1/C2); hexokinase 2; hexosaminidase A (alpha polypeptide); HHIP-like 1; HHIP-like 2; high mobility group AT-hook 2; HIR histone cell cycle regulation defective homolog A (S. cerevisiae); histone cluster 1, H2bk; histone cluster 1, H2bl; histone cluster 1, H3g; HKR1, GLI-Kruppel zinc finger family member; homeobox A7; homeobox B9;
homeodomain interacting protein kinase 2; homeodomain interacting protein kinase 3; homer homolog 2 (Drosophila); HOP homeobox; HORMA domain containing 2;
hyaluronoglucosaminidase 1 ; hydrocephalus inducing homolog (mouse); hydroxysteroid (17- beta) dehydrogenase 12; hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor); IKAROS family zinc finger 3 (Aiolos); IKBKB interacting protein;
immunoglobulin superfamily containing leucine-rich repeat 2; importin 5; ΓΝΟ80 homolog (S. cerevisiae); inositol 1,4,5-triphosphate receptor, type 3; inositol hexakisphosphate kinase 2; inositol monophosphatase domain containing 1 ; insulin-like growth factor 1 (somatomedin C); insulin-like growth factor 2 mRNA binding protein 2; insulin-like growth factor 2 mRNA binding protein 3; integrin, alpha D; integrin, alpha E (antigen CD 103, human mucosal lymphocyte antigen 1 ; alpha polypeptide); interleukin 1 receptor, type II; interleukin 32;
interleukin 4 receptor; interleukin- 1 receptor-associated kinase 2; internexin neuronal intermediate filament protein, alpha; intracisternal A particle-promoted polypeptide; IQ motif and Sec7 domain 1 ; IQ motif and Sec7 domain 2; IQ motif and Sec7 domain 3; IQ motif containing H; iroquois homeobox 5; isocitrate dehydrogenase 2 (NADP+), mitochondrial; jagged 1 (Alagille syndrome); janus kinase and microtubule interacting protein 2; junctophilin 4; kelch repeat and BTB (POZ) domain containing 7; keratin 222; keratin 73; keratin 82; KH domain containing, RNA binding, signal transduction associated 3; KIAAOlOl ; KIAA0174; KIAA0182; KIAA0240; KIAA0355; KIAA0415; KIAA0913; KIAA1328; KIAA1609; KIAA1671 ;
KIAA1737; killer cell lectin-like receptor subfamily C, member 4; kinesin family member 13B; kinesin family member 18B; kinesin family member 23; kinesin family member 26B; Kv channel interacting protein 1 ; Kv channel interacting protein 2; 1(3 )mbt- like 4 (Drosophila); lactamase, beta 2; lactate dehydrogenase D; laminin, alpha 1 ; latrophilin 1 ; Leber congenital amaurosis 5; lectin, galactoside-binding, soluble, 14; lemur tyrosine kinase 3; Leol, Pafl/RNA polymerase II complex component, homolog (S. cerevisiae); leucine aminopeptidase 3; leucine carboxyl methyltransferase 1 ; leucine rich repeat and fibronectin type III domain containing 5; leucine rich repeat and Ig domain containing 1 ; leucine rich repeat and sterile alpha motif containing 1 ; leucine rich repeat containing 37, member A2; leucine rich repeat containing 37, member A4 (pseudogene); leucine rich repeat containing 37A; leucine rich repeat containing 61 ; leucine rich repeat containing 68; leucine rich repeat containing 8 family, member A; leucine- rich repeat kinase 1 ; leucine-rich repeats and calponin homology (CH) domain containing 3; leucine-rich repeats and immunoglobulin-like domains 2; leukocyte cell derived chemotaxin 1; LIM domain and actin binding 1; LIM domain binding 2; LIM domain only 1 (rhombotin 1); LIM homeobox 3; lin-37 homolog (C. elegans); lipase maturation factor 1 ; lipase, hepatic; lipocalin 8; low density lipoprotein receptor class A domain containing 1 ; low density lipoprotein receptor-related protein 4; lymphocyte antigen 6 complex, locus E; lymphocyte antigen 9; LYR motif containing 4; lysophospholipase I; lysosomal protein transmembrane 4 beta; lysyl oxidase-like 1; mahogunin, ring finger 1; major facilitator superfamily domain containing 1 1; major histocompatibility complex, class I, A; major histocompatibility complex, class II, DM alpha; major histocompatibility complex, class II, DM beta; major vault protein; makorin ring finger protein 2; mannosyl (beta- 1 ,4-)-glycoprotein beta-l,4-N- acetylglucosaminyltransferase; MAP7 domain containing 2; mastermind-like 3 (Drosophila); MAX dimerization protein 1; Mdm2 p53 binding protein homolog (mouse); mediator complex subunit 13 -like; mediator complex subunit 24; mediator complex subunit 27; mediator complex subunit 7; melanoma antigen family D, 1; mesoderm specific transcript homolog (mouse);
metastasis associated 1 ; metastasis associated 1 family, member 3; metastasis associated in colon cancer 1 ; methionine adenosyltransferase II, beta; methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2-like; methyltransferase like 14; methyltransferase like 2B;
methyltransferase like 8; methyltransferase like 9; mevalonate (diphospho) decarboxylase; microcephalin 1 ; microsomal glutathione S-transferase 2; microsomal glutathione S-transferase 3; microtubule associated serine/threonine kinase 2; microtubule associated serine/threonine kinase family member 4; microtubule associated tumor suppressor candidate 2; microtubule- associated protein 1 light chain 3 beta 2; mindbomb homolog 2 (Drosophila); mitochondrial poly(A) polymerase; mitochondrial ribosomal protein L14; mitochondrial ribosomal protein L21 ; mitochondrial ribosomal protein L39; mitochondrial ribosomal protein L46; mitochondrial ribosomal protein S28; mitochondrial transcription termination factor; mitochondrial translational release factor 1 ; mitogen-activated protein kinase 15; mitogen-activated protein kinase binding protein 1 ; mitogen-activated protein kinase kinase kinase 14; mitogen-activated protein kinase kinase kinase 7; MLX interacting protein; MOB1, Mps One Binder kinase activator-like IB (yeast); MO 1 homolog B (yeast); MON2 homolog (S. cerevisiae); monocyte to macrophage differentiation-associated; MORN repeat containing 4; mortality factor 4 like 2; musashi homolog 2 (Drosophila); muscle, skeletal, receptor tyrosine kinase; muskelin 1, intracellular mediator containing kelch motifs; myelodysplasia syndrome 2 translocation associated; myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila);
myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 1; myeloid/lymphoid or mixed-lineage leukemia 2; myosin ID; myosin VB; myosin VC; myosin VIIA and Rab interacting protein; myosin VIIB; myosin, heavy chain 3, skeletal muscle, embryonic; myosin, light chain 12A, regulatory, non-sarcomeric; myosin, light chain 4, alkali; atrial, embryonic; myozenin 3; NACC family member 2, BEN and BTB (POZ) domain containing; N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase); N- acetyltransferase 1 (arylamine N-acetyltransferase); NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa; NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7, 14.5kDa; NADH dehydrogenase (ubiquinone) Fe-S protein 4, 18kDa (NADH-coenzyme Q reductase); naked cuticle homolog 2 (Drosophila); NCK adaptor protein 2; neighbor of BRCA1 gene 1; neogenin homolog 1 (chicken); N-ethylmaleimide-sensitive factor attachment protein, alpha; neural precursor cell expressed, developmentally down-regulated 9; neurensin 1 ; neurensin 2; neuritin 1; neuroblastoma breakpoint family, member 16; neuroblastoma breakpoint family, member 9; neuroblastoma, suppression of tumorigenicity 1; neurocalcin delta;
neurocanthocytosis; neurofibromin 1 ; neurogenic differentiation 6; neuroligin 4, X-linked;
neuromedin U receptor 1; neuronal PAS domain protein 1; NIMA (never in mitosis gene a)- related kinase 3; NK3 homeobox 2; non-SMC element 2, MMS21 homolog (S. cerevisiae); Notch homolog 2 (Drosophila); N-terminal EF-hand calcium binding protein 1 ; nuclear factor I/A; nuclear fragile X mental retardation protein interacting protein 1 ; nuclear mitotic apparatus protein 1 ; nuclear pore complex interacting protein-like 1 ; nuclear pore complex interacting protein-like 3; nuclear prelamin A recognition factor-like; nuclear receptor coactivator 2; nuclear receptor coactivator 3; nuclear receptor corepressor 1 ; nuclear receptor subfamily 2, group F, member 2; nuclear undecaprenyl pyrophosphate synthase 1 homolog (S. cerevisiae);
nucleobindin 2; nucleolar protein 10; nucleoporin 107kDa; nucleoporin 188kDa; nucleoporin 214kDa; NudC domain containing 1 ; nudix (nucleoside diphosphate linked moiety X)-type motif 13; numb homolog (Drosophila); nurim (nuclear envelope membrane protein); olfactomedin-like 1; olfactory receptor, family 1, subfamily J, member 1 ; olfactory receptor, family 11, subfamily H, member 2; olfactory receptor, family 4, subfamily F, member 29; olfactory receptor, family 7, subfamily E, member 8 pseudogene; oncomodulin 2; Opa interacting protein 5; Opa interacting protein 5; orofacial cleft 1 candidate 1; otopetrin 1 ; OTU domain containing 7A; PAN3 poly(A) specific ribonuclease subunit homolog (S. cerevisiae); pancreatic and duodenal homeobox 1; papilin, proteoglycan-like sulfated glycoprotein; patched domain containing 1 ; patched homolog
1 (Drosophila); PBX/knotted 1 homeobox 1; PCF11, cleavage and polyadenylation factor subunit, homolog (S. cerevisiae); PDS5, regulator of cohesion maintenance, homolog A (S. cerevisiae); PDZ and LIM domain 2 (mystique); PDZ and LIM domain 7 (enigma); PDZ domain containing ring finger 3; peptidylprolyl isomerase A (cyclophilin A)-like 4A; peripherin;
peroxiredoxin 6; peroxisome proliferator-activated receptor gamma, coactivator 1 beta; PH domain and leucine rich repeat protein phosphatase 1 ; PHD and ring finger domains 1 ; PHD finger protein 12; PHD finger protein 13; PHD finger protein 17; phosphatase and tensin homolog; phosphatidic acid phosphatase type 2B; phosphatidylethanolamine-binding protein 4; phosphatidylinositol binding clathrin assembly protein; phosphatidylinositol glycan anchor biosynthesis, class B; phosphatidylinositol transfer protein, membrane-associated 2;
phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1; phosphatidylinositol- 4-phosphate 5-kinase, type I, beta; phosphodiesterase 3A, cGMP-inhibited; phosphoenolpyruvate carboxykinase 1 (soluble); phosphoglucomutase 2-like 1 ; phosphoglucomutase 5;
phosphoinositide-3-kinase, class 2, alpha polypeptide; phosphoinositide-3-kinase, class 3;
phospholipase A2, group IVC (cytosolic, calcium-independent); phospholipase A2, group IVE; phosphoprotein associated with glycosphingolipid microdomains 1 ; phosphorylase kinase, alpha
2 (liver); phosphorylase, glycogen, liver; pituitary tumor-transforming 1 interacting protein; plasminogen-like B2; platelet-activating factor acetylhydrolase lb, regulatory subunit 1 (45kDa); pleckstrin homology domain containing, family G (with RhoGef domain) member 3 ;
pleomorphic adenoma gene-like 1 ; plexin CI ; PMS1 postmeiotic segregation increased 1 (S. cerevisiae); PNMA-like 1 ; poliovirus receptor-related 1 (herpesvirus entry mediator C); polo-like kinase 1 (Drosophila); poly(A) binding protein, cytoplasmic 4 (inducible form); poly(rC) binding protein 1; polybromo 1 ; polycystic kidney disease (polycystin) and REJ homolog (sperm receptor for egg jelly homolog, sea urchin); polymerase (RNA) I polypeptide A, 194kDa; polymerase (RNA) III (DNA directed) polypeptide B; post-GPI attachment to proteins 2;
potassium channel tetramerisation domain containing 21 ; potassium channel tetramerisation domain containing 7; potassium channel, subfamily T, member 2; potassium inwardly-rectifying channel, subfamily J, member 16; potassium voltage-gated channel, delayed-rectifier, subfamily S, member 1 ; potassium voltage-gated channel, KQT-like subfamily, member 2; potassium voltage-gated channel, Shaw-related subfamily, member 2; PPPDE peptidase domain containing 2; PR domain containing 11 ; PR domain containing 14; PR domain containing 4; prickle homolog 1 (Drosophila); primase, DNA, polypeptide 2 (58kDa); programmed cell death 7; prolactin; proline rich 14; proline rich 7 (synaptic); prolyl 4-hydroxylase, alpha polypeptide I; proprotein convertase subtilisin/kexin type 6; prostate stem cell antigen; prostate tumor overexpressed 1 ; protease, serine, 12 (neurotrypsin, motopsin); proteasomal ATPase-associated factor 1 ; proteasome (prosome, macropain) 26S subunit, non-ATPase, 1 1; proteasome (prosome, macropain) 26S subunit, non-ATPase, 7; proteasome (prosome, macropain) activator subunit 4; proteasome (prosome, macropain) subunit, alpha type, 5; proteasome (prosome, macropain) subunit, beta type, 6; protein arginine methyltransferase 8; protein geranylgeranyltransferase type I, beta subunit; protein inhibitor of activated STAT, 1 ; protein kinase C, beta; protein kinase C, eta; protein kinase D2; protein phosphatase 1, regulatory (inhibitor) subunit 16A; protein phosphatase 1, regulatory (inhibitor) subunit 3G; protein phosphatase 2, regulatory subunit B", gamma; protein phosphatase, Mg2+/Mn2+ dependent, IB; protein serine kinase H2; protein tyrosine phosphatase type IVA, member 3; protein tyrosine phosphatase, non-receptor type 20A; protein tyrosine phosphatase, receptor type, B; protein tyrosine phosphatase, receptor type, S; protocadherin 1 ; protocadherin alpha 12; protocadherin alpha 3; protocadherin beta 14;
protocadherin beta 15; protocadherin beta 18 pseudogene; protocadherin beta 3; protocadherin gamma subfamily C, 3; PRP39 pre-mRNA processing factor 39 homolog (S. cerevisiae);
pseudouridylate synthase 10; PTPRF interacting protein, binding protein 1 (liprin beta 1);
purinergic receptor P2X, ligand-gated ion channel, 4; purine-rich element binding protein A; pyrroline-5-carboxylate reductase 1 ; quaking homolog, KH domain RNA binding (mouse); quiescin Q6 sulfhydryl oxidase 1 ; RAB GTPase activating protein 1; RAB27B, member RAS oncogene family; RAB2A, member RAS oncogene family; RAB3A interacting protein (rabin3); RAB6C, member RAS oncogene family; RAB7, member RAS oncogene family-like 1; rabaptin, RAB GTPase binding effector protein 1 ; RADl homolog (S. pombe); RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae); RAD52 motif 1 ; Rap guanine nucleotide exchange factor (GEF) 3; RAPl interacting factor homolog (yeast); RAR-related orphan receptor A; Ras association (RalGDS/AF-6) domain family (N-terminal) member 10; Ras association
(RalGDS/AF-6) domain family (N-terminal) member 8; ras homolog gene family, member Q; RAS p21 protein activator 4; RAS protein activator like 2; ras responsive element binding protein 1 ; RCSD domain containing 1 ; receptor-interacting serine-threonine kinase 2; RecQ protein-like 5; regulator of chromosome condensation 1; regulator of G-protein signaling 20; regulator of G-protein signaling 7; regulatory associated protein of MTOR, complex 1;
regulatory factor X, 3 (influences HLA class II expression); reticulocalbin 1, EF-hand calcium binding domain; reticulon 2; reticulon 4; retinoic acid receptor, alpha; retinol dehydrogenase 13 (all-trans/9-cis); retrotransposon gag domain containing 4; REX1, RNA exonuclease 1 homolog (S. cerevisiae); REXl, RNA exonuclease 1 homolog (S. cerevisiae)-like 2 (pseudogene);
rhabdoid tumor deletion region gene 1 ; Rho GTPase activating protein 1 1A; Rho GTPase activating protein 1 IB; Rho GTPase activating protein 22; Rho guanine nucleotide exchange factor (GEF) 10-like; rhomboid 5 homolog 2 (Drosophila); rhophilin, Rho GTPase binding protein 1; Rho-related BTB domain containing 2; rhotekin 2; Rhox homeobox family, member 2; ribosomal LI domain containing 1 ; ribosomal L24 domain containing 1 ; ribosomal protein L30; ribosomal protein S7; ribosomal protein SA pseudogene 58; ribosomal protein, large, PI ; ring finger protein 139; ring finger protein 145; ring finger protein 152; ring finger protein 157; ring finger protein 165; ring finger protein 220; ring finger protein 24; ring finger protein 43; RNA binding motif protein 19; RNA binding motif protein 26; RNA binding protein SI, serine-rich domain; RNA binding protein with multiple splicing; RNA terminal phosphate cyclase domain 1; RODl regulator of differentiation 1 (S. pombe); roundabout, axon guidance receptor, homolog 3 (Drosophila); RPGRIP 1 -like; RRS1 ribosome biogenesis regulator homolog (S. cerevisiae); RUN and FYVE domain containing 1; RUN and FYVE domain containing 3; SI 00 calcium binding protein A2; saccharopine dehydrogenase (putative); sal-like 3 (Drosophila); SAM and SH3 domain containing 1; SAP30-like; sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein); sarcosine dehydrogenase; SATB homeobox 1; scaffold attachment factor B2; schlafen family member 5; scinderin; SCYl-like 1 (S. cerevisiae); Sec23 homolog B (S.
cerevisiae); secretagogin, EF-hand calcium binding protein; secreted protein, acidic, cysteine- rich (osteonectin); secretory carrier membrane protein 5; sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B; septin 9; serine hydroxymethyltransferase 1 (soluble); serine racemase;
serine/arginine repetitive matrix 3; serine/threonine kinase 17b; serine/threonine kinase 24 (STE20 homolog, yeast); serpin peptidase inhibitor, clade B (ovalbumin), member 9; SERPINEl mRNA binding protein 1 ; sestrin 3; SET and MYND domain containing 3; SET binding factor 2; SET binding protein 1 ; SET domain containing 6; seven in absentia homolog 1 (Drosophila); sex comb on midleg homolog 1 (Drosophila); SH3 and PX domains 2A; SH3 domain containing 19; SH3 -binding domain kinase 1; SHC (Src homology 2 domain containing) family, member 4; Shwachman-Bodian-Diamond syndrome; sialidase 3 (membrane sialidase); sideroflexin 5; signal peptide, CUB domain, EGF-like 3; signal recognition particle 54kDa; signal-induced
proliferation-associated 1 like 1 ; SIK family kinase 3; sine oculis binding protein homolog (Drosophila); single-stranded DNA binding protein 2; SMAD family member 3; SMAD family member 6; small EDRK-rich factor 1A (telomeric); SMEK homolog 1, suppressor of mekl (Dictyostelium); Smg-5 homolog, nonsense mediated mRNA decay factor (C. elegans);
smoothened homolog (Drosophila); snail homolog 3 (Drosophila); solute carrier family 14 (urea transporter), member 2; solute carrier family 16, member 3 (monocarboxylic acid transporter 4); solute carrier family 16, member 4 (monocarboxylic acid transporter 5); solute carrier family 2 (facilitated glucose transporter), member 11 ; solute carrier family 22, member 23; solute carrier family 25 (mitochondrial carrier; peroxisomal membrane protein, 34kDa), member 17; solute carrier family 25, member 13 (citrin); solute carrier family 25, member 30; solute carrier family 26, member 10; solute carrier family 35, member F5; solute carrier family 37 (glycerol-3- phosphate transporter), member 3; solute carrier family 39 (metal ion transporter), member 11 ; solute carrier family 39 (zinc transporter), member 14; solute carrier family 39 (zinc transporter), member 2; solute carrier family 46, member 2; solute carrier family 6 (neurotransmitter transporter, taurine), member 6; solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 ; sorbin and SH3 domain containing 3; sortilin-related receptor, L(DLR class) A repeats-containing; sorting nexin 20; spastic paraplegia 7 (pure and complicated autosomal recessive); spermatogenesis associated 13; spermatogenesis associated 5-like 1 ; spinster homolog 3 (Drosophila); spleen focus forming virus (SFFV) proviral integration oncogene spil ; spleen tyrosine kinase; splicing factor, arginine/serine-rich 1; splicing factor, arginine/serine-rich 14; splicing factor, arginine/serine-rich 8 (suppressor-of-white-apricot homolog, Drosophila); sprouty-related, EVH1 domain containing 1 ; SPT2, Suppressor of Ty, domain containing 1 (S. cerevisiae); Src homology 2 domain containing adaptor protein B; SREBF chaperone; SRY (sex determining region Y)-box 12; SRY (sex determining region Y)-box 21 ; SRY (sex determining region Y)-box 7; ST3 beta-galactoside alpha-2,3-sialyltransferase 4; ST8 alpha-N-acetyl- neuraminide alpha-2,8-sialyltransferase 2; stanniocalcin 2; staufen, RNA binding protein, homolog 2 (Drosophila); STE20-related kinase adaptor alpha; sterile alpha motif domain containing 4A; storkhead box 2; strawberry notch homolog 2 (Drosophila); striatin, calmodulin binding protein 3; stromal interaction molecule 1 ; structural maintenance of chromosomes 2; SUMOl/sentrin specific peptidase 6; supervillin; suppressor of cytokine signaling 2; sushi domain containing 1; SV2 related protein homolog (rat); synapsin III; synaptic vesicle glycoprotein 2B; synaptonemal complex protein 2-like; synaptosomal-associated protein, 47kDa; synaptotagmin I; synaptotagmin II; synaptotagmin V; syntrophin, beta 1 (dystrophin-associated protein Al, 59kDa, basic component 1); synuclein, beta; T cell receptor alpha constant; T cell receptor beta variable 30 (gene/pseudogene); T cell receptor gamma variable 9; TAF 15 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 68kDa; talin 2; tankyrase, TRF 1 -interacting ankyrin-related ADP-ribose polymerase; tankyrase, TRF 1 -interacting ankyrin- related ADP-ribose polymerase 2; TAO kinase 3; tau tubulin kinase 2; TBC1 domain family, member 16; TBC1 domain family, member 3C; TBC1 domain family, member 8 (with GRAM domain); tectonin beta-propeller repeat containing 2; tensin like CI domain containing phosphatase (tensin 2); tetraspanin 10; tetraspanin 4; tetratricopeptide repeat domain 22;
tetratricopeptide repeat domain 30A; tetratricopeptide repeat domain 7B; tetratricopeptide repeat domain 8; tetratricopeptide repeat domain 9; thioredoxin-related transmembrane protein 3;
thrombospondin 2; thrombospondin 4; thymopoietin; thyroid hormone receptor, beta
(erythroblastic leukemia viral (v-erb-a) oncogene homolog 2, avian); tigger transposable element derived 4; tigger transposable element derived 6; tight junction protein 2 (zona occludens 2); tight junction protein 3 (zona occludens 3); TIMP metallopeptidase inhibitor 3; toll interacting protein; toll-like receptor 6; tolloid-like 1; torsin A interacting protein 1 ; torsin A interacting protein 2; tousled-like kinase 2; TOX high mobility group box family member 3; TP53RK binding protein; TRAF3 interacting protein 2; trafficking protein particle complex 9;
transcription factor 12; transcription factor 4; transcription factor 7-like 1 (T-cell specific, HMG- box); transcription factor AP-4 (activating enhancer binding protein 4); transcription factor Dp-1; transcription factor Dp-2 (E2F dimerization partner 2); transducin (beta)-like 1 X-linked receptor 1; transducin-like enhancer of split 3 (E(spl) homolog, Drosophila); transferrin receptor (p90, CD71); transient receptor potential cation channel, subfamily A, member 1 ; transmembrane 6 superfamily member 2; transmembrane 7 superfamily member 3; transmembrane 9 superfamily member 2; transmembrane and coiled-coil domain family 1; transmembrane and coiled-coil domains 4; transmembrane and ubiquitin-like domain containing 2; transmembrane emp24 domain trafficking protein 2; transmembrane protein 131 ; transmembrane protein 132C;
transmembrane protein 132D; transmembrane protein 135; transmembrane protein 14B;
transmembrane protein 164; transmembrane protein 17; transmembrane protein 180;
transmembrane protein 181; transmembrane protein 183A; transmembrane protein 188;
transmembrane protein 219; transmembrane protein 233; transmembrane protein 45B;
transmembrane protein 59; transmembrane protein 64; transmembrane protein 66;
transmembrane protein 66; transmembrane protein 86B; transthyretin; trichohyalin-like 1 ;
trinucleotide repeat containing 18; trinucleotide repeat containing 6A; tripartite motif-containing 16-like; tripartite motif-containing 27; tripartite motif-containing 54; tripartite motif-containing 69; tripartite motif-containing 9; TRM2 tRNA methyltransferase 2 homolog B (S. cerevisiae); trophinin associated protein (tastin); tropomyosin 3; tubby homolog (mouse); tuberous sclerosis 1 ; tubulin, beta 2B; tumor necrosis factor receptor superfamily, member 10c, decoy without an intracellular domain; tumor necrosis factor receptor superfamily, member 1A; tweety homolog 2 (Drosophila); tyrosylprotein sulfotransferase 1 ; ubiquinol-cytochrome c reductase, Rieske iron- sulfur polypeptide 1; ubiquitin C; ubiquitin protein ligase E3 component n-recognin 3 (putative); ubiquitin protein ligase E3 component n-recognin 5; ubiquitin specific peptidase 10; ubiquitin specific peptidase 15; ubiquitin specific peptidase 29; ubiquitin specific peptidase 8; ubiquitin- conjugating enzyme E2D 3 (UBC4/5 homolog, yeast); ubiquitin-conjugating enzyme E2G 1 (UBC7 homolog, yeast); U-box domain containing 5; UDP-N-acetyl-alpha-D- galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7); UDP-N-acetyl- alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 4; UDP-N-acetyl- alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 5; unc-93 homolog Bl (C. elegans); uveal autoantigen with coiled-coil domains and ankyrin repeats; vaccinia related kinase 2; vaccinia related kinase 3; vacuolar protein sorting 13 homolog A (S. cerevisiae);
vacuolar protein sorting 24 homolog (S. cerevisiae); vestigial like 4 (Drosophila); vinculin; vitamin K epoxide reductase complex, subunit 1 -like 1 ; v-myc myelocytomatosis viral oncogene homolog (avian); Vpr (HIV-1) binding protein; v-ral simian leukemia viral oncogene homolog B (ras related; GTP binding protein); v-ski sarcoma viral oncogene homolog (avian); WAS protein family homolog 2 pseudogene; WD repeat domain 1 ; WD repeat domain 20; WD repeat domain 27; WD repeat domain 31 ; WD repeat domain 44; WD repeat domain 70; WD repeat domain 85; Werner helicase interacting protein 1; Williams-Beuren syndrome chromosome region 16;
wingless-type MMTV integration site family, member 3 A; WNT1 inducible signaling pathway protein 3 ; WW domain binding protein 1 1 ; WWC family member 3 ; X-linked inhibitor of apoptosis; XRCC6 binding protein 1; YTH domain family, member 1 ; zinc finger and AT hook domain containing; zinc finger and BTB domain containing 45; zinc finger and BTB domain containing 9; zinc finger and SCAN domain containing 22; zinc finger CCCH-type, antiviral 1; zinc finger E-box binding homeobox 1 ; zinc finger protein 100; zinc finger protein 114; zinc finger protein 121; zinc finger protein 160; zinc finger protein 161 homolog (mouse); zinc finger protein 19; zinc finger protein 192; zinc finger protein 205; zinc finger protein 213; zinc finger protein 254; zinc finger protein 256; zinc finger protein 263; zinc finger protein 283; zinc finger protein 30; zinc finger protein 318; zinc finger protein 322B; zinc finger protein 329; zinc finger protein 354B; zinc finger protein 416; zinc finger protein 426; zinc finger protein 429; zinc finger protein 432; zinc finger protein 44; zinc finger protein 445; zinc finger protein 45; zinc finger protein 454; zinc finger protein 461 ; zinc finger protein 468; zinc finger protein 470; zinc finger protein 484; zinc finger protein 490; zinc finger protein 507; zinc finger protein 516; zinc finger protein 521; zinc finger protein 528; zinc finger protein 529; zinc finger protein 562; zinc finger protein 572; zinc finger protein 583; zinc finger protein 584; zinc finger protein 587; zinc finger protein 605; zinc finger protein 619; zinc finger protein 62 homolog (mouse); zinc finger protein 642; zinc finger protein 655; zinc finger protein 667; zinc finger protein 679; zinc finger protein 681; zinc finger protein 697; zinc finger protein 705E; zinc finger protein 706; zinc finger protein 707; zinc finger protein 710; zinc finger protein 717; zinc finger protein 730; zinc finger protein 747; zinc finger protein 749; zinc finger protein 764; zinc finger protein 787; zinc finger protein 790; zinc finger protein 8; zinc finger protein 808; zinc finger protein 814; zinc finger protein 816A; zinc finger protein 835; zinc finger protein 84; zinc finger protein 841 ; zinc finger protein 85; zinc finger protein 90; zinc finger protein 92; zinc finger protein, multitype 2; zinc finger, AN 1 -type domain 1; zinc finger, BED-type containing 5; zinc finger, CCHC domain containing 13; zinc finger, DHHC-type containing 2; zinc finger, FYVE domain containing 20; zinc finger, HIT type 6; zinc finger, imprinted 2; zinc finger, SWIM-type containing 5; zinc finger, X-linked, duplicated A; zinc fingers and homeoboxes 2; and zymogen granule protein 16 homolog (rat).
[0034] It is expected that modulation or alteration of IncRNA function or levels can be used to regulate chromatin status, gene expression, transcription, translation, post-translational events and global biomolecular trafficking in the cell, especially to and from the nucleus.
[0035] Methods of designing, modulating or targeting IncRNAs may be either structure-based or sequence based. Traditionally, methods of targeting nucleic acid molecules in the cell have been sequenced based and have depended in some form on harnessing the hybridization or base pairing of two complementary molecules. Sequence-based methods of modulating or altering IncRNA function and levels are described herein.
[0036] Also described are herein structure-based methods. As used herein, "structure based methods" are those methods of altering or modulating a IncRNA function or level that depends on the determination or knowledge of the higher order structure of at least a portion of a IncRNA target. "Higher order structures" include but are not limited to the overall secondary, tertiary or quarternary structure of a molecule, e.g., hairpin structures, bulges, etc. These structures may be determined informatically with prediction algorithms based on thermodynamic parameters and energy calculations. Preferably, secondary structure prediction is performed using either M-fold or RNA Structure algorithm. Programs for secondary structure determination are freely available online. Structures may also be determined by NMR, Mass Spectroscopy or by crystallographic methods. For RNA molecules, methods of determining overall structure or structures of portions of the RNA molecule are known in the art. [0037] In US Patent 6,221,587, incorporated herein by reference in its entirety, are methods of identifying secondary structures in eukaryotic and prokaryotic RNA molecules termed "molecular interaction sites." Molecular interaction sites are small, usually less than 30 nucleotides, independently folded, functional subdomains contained within a larger RNA molecule. These methods may be used to determine molecular interaction sites on IncRNAs.
[0038] IncRNA targets may also be subjected to mimicry design. Disclosed in US Patent 6,368,863 incorporated herein by reference in its entirety, are methods of identifying protein interacting sites on an RNA molecule and then designing an oligonucleotide that mimics that portion of the larger RNA molecules. These methods may be used in the present invention to design small IncRNA target mimics which will bind proteins.
[0039] Unlike sequence-based or hybridization driven targeting, which must rely on access of the targeting molecule to the target in order for base pairing to occur, structure-based targeting embraces a larger portion of the IncRNA target.
[0040] "Features" when referring to IncRNAs are defined as distinct nucleic acid-based components of the molecule. Features of the IncRNAs of the present invention may be structural features and may include surface manifestations, local conformational shape, folds, loops, half- loops, domains, half-domains, sites, termini or any combination thereof. When designing IncRNA variant molecules, the starting molecule may be one selected from Table 2 or known in the cell as the wild type molecule. Alternatively, a series of modifications may be made in which the starting molecule may be referred to simply as the parent molecule. Structural features of the present invention may be at least 200 nucleotides in length or from about 200 to about 500 nucleotides in length or from about 200 to about 300 nucleotides in length or from about 50 to about 100 nucleotides in length. They may also comprise the whole or any part of a defined structural feature. Structural features may be 4-10, 5-15, 10-20, 10-30, or 20-50 nucleotides in length. These may be represented in increments of the triplet code and therefore may be any multiple of three. For example, features may be from 15-18, 15-30, 15-36, 15-60, 30-60, 30-90, 30-120 or larger.
[0041] As used herein when referring to IncRNAs the term "surface manifestation" refers to a nucleic acid based component of a IncRNA appearing on an outermost surface of the IncRNA.
[0042] As used herein when referring to IncRNAs the term "local conformational shape" means a nucleic acid based structural manifestation of a IncRNA which is located within a definable space of the IncRNA.
[0043] As used herein when referring to IncRNAs the term "fold" means the resultant conformation of a nucleic acid sequence upon energy minimization. A fold may occur at the secondary or tertiary level of the folding process. Examples of secondary level folds include hairpins, loops and bulges. Examples of tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include
hydrophobic and hydrophilic pockets, and the like.
[0044] As used herein the term "turn" as it relates to IncRNA conformation means a bend which alters the direction of the backbone of a poly- or oligonucleotide and may involve one, two, three or more nucleotides.
[0045] As used herein when referring to IncRNAs the term "loop" refers to a structural feature of a poly- or oligonucleotide which reverses the direction of the backbone of a poly- or oligonucleotide and comprises four or more nucleotides.
[0046] As used herein when referring to IncRNAs the term "half-loop" refers to a portion of an identified loop having at least half the number of nucleotides as the loop from which it is derived. It is understood that loops may not always contain an even number of nucleotides. Therefore, in those cases where a loop contains or is identified to comprise an odd number of nucleotides, a half-loop of the odd-numbered loop will comprise the whole number portion or next whole number portion of the loop (number of nucleotides of the loop/2+/-0.5 nucleotides). For example, a loop identified as a 7 nucleotide loop could produce half-loops of 3 nucleotides or 4 nucleotides (7/2=3.5+/-0.5 being 3 or 4).
[0047] As used herein when referring to IncRNAs the term "domain" refers to a motif of a poly- or oligonucleotide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions, etc).
[0048] As used herein when referring to IncRNAs the term "half-domain" means portion of an identified domain having at least half the number of nucleotides as the domain from which it is derived. It is understood that domains may not always contain an even number of nucleotides. Therefore, in those cases where a domain contains or is identified to comprise an odd number of nucleotides, a half-domain of the odd-numbered domain will comprise the whole number portion or next whole number portion of the domain (number of nucleotides of the domain/2+/-0.5 nucleotides). It is also understood that sub-domains may be identified within domains or half- domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the nucleotides that comprise any of the domain types herein need not be contiguous along the backbone of the poly- or oligonucleotide (i.e., nonadjacent nucleotides may fold structurally to produce a domain, half-domain or subdomain).
[0049] As used herein when referring to IncRNAs the term "site" represents a location for targeting a IncRNA. A site represents a position within a poly- or oligonucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention. In one embodiment, "sites" of targeting can represent hundreds to thousands of nucleotides and may include nucleotides very distal in sequence location. For example, upon folding, IncRNAs may present surfaces, domains or sites which comprise nucleotides which ony appear juxtaposed due to the folded nature of the IncRNA. In one embodiment of the invention, LDAs may target any site on a IncRNA.
[0050] As used herein the terms "termini or terminus" when referring to IncRNAs refers to an extremity of a poly- or oligonucleotide. Such extremity is not limited only to the first or final site of the poly- or oligonucleotide but may include additional nucleotides in the terminal regions. The poly- or oligonucleotide based molecules of the present invention may be characterized as having both a 5' and a 3 ' terminus. Poly- or oligonucleotides of the invention are in some cases made up of multiple chains brought together by disulfide bonds or by non- covalent forces (multimers, oligomers or dendrimers). These sorts of poly- or oligonucleotides will have multiple 5' and 3 '-termini. Alternatively, the termini of the poly- or oligonucleotide may be modified such that they begin or end, as the case may be, with a non- poly- or oligonucleotide based moiety such as a conjugate.
[0051] Once any of the features have been identified or defined as a component of a IncRNA of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating them to produce additional IncRNA variants.
[0052] In one embodiment a IncRNA transcript variant is designed to encode a LDA. For example, a IncRNA transcript RNA molecule may comprise the sequence of a shRNA or other nucleic acid based LDA. When encoded in the IncRNA transcript variant the LDA (nucleic acid based) may be one that targets a different site on the IncRNA in which is is encoded or it may target the RNA transcript of a coding gene or any nucleic acid based transcript to which it will either hybridize (sequence based targeting) or form an interation with (structure based targeting).
[0053] Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.
[0054] Modifications and manipulations can be accomplished by methods known in the art. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
[0055] In one embodiment of the invention, a feature of a IncRNA transcript is removed to produce a IncRNA transcript variant. [0056] In one embodiment of the invention, a feature of a IncRNA transcript is duplicated to produce a IncRNA transcript variant.
[0057] In one embodiment of the invention, a feature of a IncRNA is swapped with a second feature of a IncRNA to produce a IncRNA variant. In some embodiments the second feature is from the same or a different IncRNA transcript.
[0058] In one embodiment, hairpin features of a IncRNA are altered or modified. Hairpin structures of a first IncRNA may be inserted into a second IncRNA. They may also be removed from a IncRNA. Where a feature is found to have a biological activity such as an interface for binding, or as a signal for localization, the feature may be reproduced in isolation by chemical or synthethic methods and used as a IncRNA directed agent of the invention.
[0059] In one embodiment of the invention, IncRNAs may be targeted to alter cellular memory, or cell identity. Without wishing to be bound by theory, it is believed that the IncRNAs may contribute to cellular memory and play a determinative role in the cells ability to produce daughter cells of the same lineage or RNA population signature thereby maintaining the identity of the cells during cell divisions. As used herein, the "RNA population signature" of a cell is the qualitative complement of RNA transcripts present in a cell at a particular time or timeframe that distinguishes the cell from other cell types. It should be understood that an RNA population signature of a cell does not necessarily comprise the sum total of all RNAs present in a cell but a set or subset of transcripts which may be used to identify one cell type from another cell type. RNA population signatures may comprise "nuclear signatures", "cytoplasmic signatures", "organelle associated signatures," "tissue-associated signatures," or combinations thereof. They may also comprise the set of IncRNA genes or transcripts or subsets thereof.
[0060] For example, the RNA nuclear population signature of a cell may comprise the set or subset of RNA transcripts present in the nucleus of the cell at a particular time or developmental phase such that this signature can be compared to other cells in order to determine whether the cells are of the same type or along the same path of lineage. Methods of measuring the presence of RNA in a cell are well known in the art. Methods of determining the cell type of a specific cell are known in the art and include for example the measurement of cell type specific markers such as proteins or protein or ligand expression, receptor or ligand presence or secretion.
[0061] In one embodiment of the present invention, the RNA population signature of a cell may be measured or identified and compared to another cell. The comparison will reveal differences between the two signatures. Differences in the IncRNA transcript components of the signature may then be assessed and a cellular RNA population may be supplemented or reduced to effect a similar signature in the target cell population. [0062] Cells of the present invention include, but are not limited to, cells that are derived primarily from endoderm (gland cells, exocrine secretory epithelial cells, hormone secreting cells, epithelial cells lining closed internal body cavities); cells derived primarily from ectoderm (integumentary system, keratinizing epithelial cells, wet stratified barrier epithelial cells, nervous system cells, sensory transducer cells, autonomic neuron cells, sense organ and peripheral neuron supporting cells, central nervous system neurons and glial cells, lens cells); cells derived primarily from mesoderm (metabolism and storage cells, barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract), kidney; extracellular matrix secretion cells, contractile cells, blood and immune system cells, pigment cells, nurse cells and interstitial cells). Stem cells include, but are not limited to adult, embryonic, pluripotent, totipotent, and induced pluripotent.
[0063] In one embodiment of the invention are methods of controlling or reprogramming the cellular memory of a cell by adminsitering to said cell or introducing into said cell a LDA which alters the function of a IncRNA. Alternatively, the RNA population signature may be altered by adding back one or more IncRNA genes or transcripts. By altering the level of IncRNA, cellular processes are altered such that the cell may differentiate along a different path to alter the phenotype of the cell or to mirror the RNA population signature of a target cell.
[0064] Alternatively, it is contemplated that the RNA population signature of a cell may be altered by administering an LDA or an exogenously prepared IncRNA gene or transcript, the outcomes of which would result in the alteration of the cellular phenotype (See Arancio, W., Rejuvenation Research, 13, 1-8, 2010). In one example, cells may be treated to alter the lineage or differentiation state of for example stem cells or cells of early developmental lineage.
[0065] These methods and compositions have utility in the areas of not only cellular regulation but in the field of stem cell technology and the guided evolution of cellular phenotypes.
[0066] In one embodiment of the invention, the RNA population signature of a cell, cell line or tissue may be used in diagnostic applications. According to the present invention, methods are provided for the use of the RNA population signature, more specifically the IncRNA population signature of a cell or tissue, in diagnostic applications. In this method, IncRNAs associated with a disease or condition or having a linkage related to chromosome location are measured and where the level of IncRNAs are not similar or identical to a normal cell of the same lineage, one or more LDAs are administered which increase or decrease the level of IncRNA as desired.
[0067] It is also the case that IncRNAs may be supplemented in cells or cell populatations that lack the normative complement of IncRNA genes or transcripts. [0068] In one embodiment, the milieu of a cell or tissue may be used to provide a signaling environment for the study of IncRNA regulation. As used herein, the term "milieu of a cell or tissue" means the supernatant or "soup" of a cell population or an extract of the cell system.
[0069] According to the present invention, a first population of cells may be incubated in the milieu of a second population of cells in order to provide an environment which alters the expression levels or RNA population signature of the first population of cells. To the milieu may be added one or more IncRNA genes, transcripts or LDAs of the present invention. As a consequence of this addition the development, differentiation or overall gene expression profile of the first population of cells may be changed. Cells which may be used to provide the incubating milieu or which may be incubated in the milieu include but are not limited to somatic or gamete, stem cells, pluripotetent cells, cells of primary origin, cells of any mammalian tissue, etc.
[0070] In one embodiment of the invention, chromatin inactivation or activation may be effected by the administration of one or more LDAs (whether sequence based or structure based) or the administration of one or more IncRNA transcripts. According to the present invention, IncRNA transcripts may be added to cells or cell systems to alter the epigenetic landscape of a cell or tissue.
[0071] In one embodiment of the invention, IncRNA cassettes may be added to or administered to a cell or tissue. As used herein a "IncRNA cassette" is a polynucleotide that encodes one or more IncRNAs. IncRNA cassettes may endcode a full length wild type IncRNA or may be designed to encode a modified IncRNA. The term "modified IncRNA" means a IncRNA which differs from the wild type sequence of the IncRNA in question. Modifications to IncRNAs include those modifications to the exonic structure of the IncRNA and include those having shuffled exon structures, omitted exons and additional exons. In all cases, by definition given that they are non-coding, a wild type IncRNA will never encode a mature protein (greater than 50 amino acids), however it is contemplated by the present invention that a IncRNA or IncRNA cassette may be designed which contains one or more complete or partial exons from a coding gene transcript. In this case, modified IncRNA transcripts could be capable of encoding a peptide or polypeptide sequence.
[0072] According to the present invention, the IncRNA cassette may also contain a modified promoter from that which is found in the wild type IncRNA. Promoters may be swapped with those of protein coding genes or other IncRNA genes. They may also be modified by addition, deletion or shuffling of promoter components. Synthetic IncRNA genes or transcripts may be modified before contacting or administration to cells and the promoters may have temporary or permanent tags or transcription factors pre-associated with them. LDAs of the present invention may also be designed to target the promoters of IncRNA genes. In this design, the LDAs may be linked to, conjutaged, associated or complexed with factors that target the LDA to the site of the IncRNA in the cell.
[0073] While not wishing to be bound by theory, it is presently understood that IncRNA transcripts do not encode mature proteins. However, it is contemplated that IncRNA transcripts may have the capacity to encode smaller proteins or peptides (Kondo, et al, Science 16 July 2010: Vol. 329. no. 5989, pp. 336 - 339). As used herein, "peptide" is an amino acid based molecule of no more than 50 amino acids.
[0074] Where IncRNA transcripts are found to encode or engineered to encode peptides, these peptides or their locus within the IncRNA transcript may be a target of the invention. Consequently, methods of regulating IncRNA transcripts of the present invention may also regulate their encoded peptides.
[0075] As used herein "synthetic" refers to a state of having been created or man-made, e.g., not of natural origin. The LDAs or IncRNAs of the present invention may be synthesized using chemical or enzymatic or recombinant methods. They may then be isolated from the synthetic mixture. Compounds of the present invention may also be isolated from a natural source.
[0076] LDAs or IncRNAs of the present invention may be associated with chromatin modifying complexes, nucleosome components, proteins or enzymes. They may also be modified to localize to either the nucleus or cytoplasm of the cells. In this manner, the LDAs or IncRNAs of the present invention may be guided to specific sites in a cell or tissue and may affect cellular processes such as gene expression, imprinting, aging, epigenetic signatures and the like.
[0077] In yet another aspect, the invention provides a method for modulating (e.g., inhibiting or activating) the expression of a IncRNA in a mammal.
[0078] Other methods of identifying ncRNAs especially IncRNAs include those described in WO/2005/060344 and WO/2003/025229 (describing efference RNA or eRNA) the contents of which are incorporated herein by reference in its entirety.
[0079] Methods of regulating IncRNAs which may act as natural antisense transcripts (NAT) are disclosed in WO/2007/087113, the contents of which are incorporated herein by refrence in its entirety. These methods may be employed in the regulation of the target ncRNA of the present invention.
II. IncRNA Directed Agents (LDAs)
[0080] The compositions of the present invention are those which may be used to regulate, control, manipulate, perturb or otherwise alter the expression, levels, activity or status of IncRNAs. As such, the compositions of the present invention are termed "IncRNA-directed agents" or "LDAs".
[0081] The LDAs of the present invention broadly include, but are not limited to, oligonucleotides, polynucleotides, iRNA agents, antisense molecules, ribozymes, aptamers, small molecules, antibodies, peptides, proteins, enzymes or fragments thereof, and vitamins.
[0082] In one embodiment, the method includes administering an LDA composition featured in the invention to the mammal such that expression of the target IncRNA is decreased, such as for an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer.
[0083] In another embodiment, the method includes administering a composition as described herein to a mammal such that expression of the target IncRNA is increased by e.g., at least 10% compared to an untreated animal. In some embodiments, the activation of IncRNA occurs over an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, four weeks, or more. Without wishing to be bound by theory, a LDA can activate IncRNA expression by stabilizing the IncRNA transcript, interacting with a promoter in the genome, and/or inhibiting an inhibitor of IncRNA expression.
[0084] Preferably, the LDAs useful for the methods and compositions featured in the invention specifically target RNAs (primary or processed) of the target IncRNA. Compositions and methods for inhibiting the expression of these IncRNAs using iRNAs can be prepared and performed as described elsewhere herein.
[0085] In one embodiment, the method includes administering a composition containing a LDA, where the LDA includes a nucleotide sequence that is complementary to at least a part of an RNA or DNA transcript of the IncRNA of the mammal to be treated. Mammals include, but are not limited to, humans, monkeys, rodents, rabbits, dogs, cats, pigs, cows, horses and the like.
[0086] The compositions and methods of the present invention find utility in research, discovery, diagnostics and therapeutic areas of human medicine, veterinary medicine, plant science and the control of pests, insects and the like.
[0087] When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection.
[0088] LDAs may be designed to target regions or sites along a IncRNA which correlate to hypersensitivity sites (HS) found on the corresponding DNA encoding the IncRNA. Hypersensitivity sites (HS) and methods of identification are described in PCT Publication WO/2004/053106, the contents of which are incorporated by reference herein in its entirety.
[0089] In one embodiment, an LDA as described herein effects inhibition IncRNA
expression. Alternatively, in another embodiment, an LDA as described herein activates IncRNA expression.
[0090] As used herein, the term "modulate the expression of," refers to an at least partial "inhibition" or partial "activation" of a IncRNA gene or transcript expression in a cell treated with a LDA composition as described herein compared to the expression of a IncRNA gene or transcript in an untreated cell. Modulation of expression may be determined not only by direct measurement of a IncRNA level after contacting with the LDA, but also be inference by associating a known phenotypic outcome which correlates to said contacting.
[0091] In one embodiment, the LDAs of the present invention may target, mimic, bind to, replace or alter the levels or function of a product of a IncRNA. As used herein "IncRNA products" include any molecule engineered to be a product of a IncRNA either by transcription, translation, cleavage, splicing, or other mechanism that produces a derivative of a IncRNA. Examples of IncRNA products include, but are not limited to, peptides or proteins engineered to be coded by the IncRNA or fragments of the IncRNA transcript.
[0092] The terms "activate," "enhance," "up-regulate the expression of," "increase the expression of," and the like, in so far as they refer to a IncRNA gene or transcript, herein refer to the at least partial activation of the expression of a IncRNA, as manifested by an increase in the amount of IncRNA transcript in whole or in part, which may be isolated from or detected in a first cell or group of cells in which a IncRNA is transcribed and which has or have been treated such that the expression of a IncRNA is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).
[0093] In one embodiment, expression of a IncRNA transcript is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a LDA as described herein. In some embodiments, a IncRNA is activated by at least about 60%, 70%, or 80% by administration of a LDA featured in the invention. In some embodiments, expression of a IncRNA transcript is activated by at least about 85%, 90%, or 95% or more by administration of a LDA as described herein. In some embodiments, the IncRNA transcript expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with a LDA as described herein compared to the expression in an untreated cell. Activation of expression of coding mRNAs by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Set U.S.A. 103 : 17337-42, and in US200701 11963 and US2005226848, each of which is incorporated herein by reference. It is believed that constructs that activate the expression of coding RNA transcripts will also activate the expression of non-coding RNA transcripts such as the lncRNA transcripts of the present invention.
[0094] The terms "silence," "inhibit the expression of," "down-regulate the expression of," "suppress the expression of," and the like, in so far as they refer to a lncRNA gene or transcript, herein refer to the at least partial suppression of the expression of lncRNA transcript, as manifested by a reduction of the amount of the lncRNA transcript in whole or in part which may be isolated from or detected in a first cell or group of cells in which a lncRNA is transcribed and which has or have been treated such that the expression of a lncRNA is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of:
[((lncRNA in control cells) - (lncRNA in treated cells))/lncRNA in control cells] xlOO %.
[0095] Alternatively, inhibition or the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to lncRNA expression, e.g., the amount of protein encoded by a mRNA that itself is controlled by a lncRNA, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production or the status of a cell, e.g., the epigenetic profile or signature of a cell which is altered upon modulation of one or more lncRNA targets.
[0096] In principle, lncRNA silencing may be determined in any cell expressing lncRNA, either constitutively or by genomic engineering, and by any appropriate assay.
[0097] For example, in certain instances, expression of a lncRNA is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a LDA featured in the invention. In some embodiments, a lncRNA is suppressed by at least about 60%, 70%, or 80% by administration of a LDA featured in the invention. In some embodiments, a lncRNA is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of a LDA as described herein.
[0098] In one embodiment of the present invention are methods of evaluating the function of IncRNAs using the cell-based assays described in US Patent Publication 20030170642, the contents of which are incorporated herein by reference in its entirety.
iRNA agents
[0099] "iRNA agents" of the present invention include, but are not limited to, small interfering RNAs (siRNA), double stranded RNAs (dsRNAs), inverted repeats, short hairpin R As (shRNAs), small temporally regulated RNAs (stR A), clustered inhibitory RNAs (cRNAs), including radial clustered inhibitory RNA, asymmetric clustered inhibitory RNA, linear clustered inhibitory RNA, and complex or compound clustered inhibitory RNA, dicer substrates, DNA-directed RNAi (ddRNAi), single-stranded R Ai (ssRNAi), microRNA (miRNA) antagonists, microRNA mimics, microRNA agonists, blockmirs (a.k.a. Xmirs), microRNA mimetics, microRNA addbacks, supermiRs, the oligomeric constructs disclosed in PCT Publication WO/2005/013901 the contents of which are incorporated herein in its entirety, tripartite RNAi constructs such as those disclosed in US Publication 20090131360, the contents of which are incorporated herein in its entirety, the solo-rxRNA constructs disclosed in PCT Publication WO/2010/01 1346, the contents of which are incorporated herein by reference in its entirety; the sd-rxRNA constructs disclosed in PCT Publication WO/2010/033247 the contents of which are incorporated herein by reference in its entirety, dual acting RNAi constructs which reduce RNA levels and also modulate the immune response as disclosed in PCT Publications WO/2010/002851 and WO/2009/141146 the contents of which are incorporated herein by reference in their entirety and antigene RNAs (agRNA) or small activiating RNAs (saRNAs) which increase expression of the target to which they are designed disclosed in PCT Publications WO/2006/130201, WO/2007/086990, WO/2009/046397, WO/2009/149182, WO/2009/086428 the contents of which are incorporated herein by reference in their entirety.
[0100] As used herein, the term "iRNA" refers to an agent that comprises at least an oligonucleotide component (e.g., nucleic acid, either RNA or DNA or modifications thereof), and which is capable of functioning through binding, preferably via hybridization. In some embodiments, the iRNA agent mediates the targeted cleavage of an RNA transcript via an RNA- induced silencing complex (RISC) pathway. In one embodiment, an iRNA agent as described herein effects inhibition IncRNA expression. Alternatively, in another embodiment, a iRNA agent as described herein activates IncRNA expression. Alternatively, in one embodiment, an iRNA agent sterically blocks access to at least a portion of the IncRNA target. Such blocking can result in the modulation of IncRNA expression, levels or function.
[0101] It is also understood that iRNA agents may act via binding but not trigger any cleavage event, but exert an effect on the function of the IncRNA target by steric means. For example, the agent may block the site of another moiety which normally would bind to the IncRNA to itself effect cleavage.
[0102] In one embodiment, the iRNA agent will comprise nucleic acid and non-nucleic acid components and the nucleic acid component may be responsible for the binding but not directly for the alteration in function of the IncRNA target. For example, conjugates of iRNA agents may have two or more functions with the nucleic acid component providing at least the hybridization function, while second, third or additional components provide functional effect to the LDA.
[0103] As used herein when referring to iRNA agents, "target sequence" refers to a contiguous portion of the nucleotide sequence of a DNA molecule of a IncRNA gene or RNA sequence formed during the transcription of a IncRNA, including the IncRNA transcript that is a product of RNA processing of a primary transcription product. Where the LDA is an iRNA agent, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed activity/ e.g., cleavage, blocking, etc) at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides,20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.
[0104] Smaller target sequences are also contemplated by the present invention. In one embodiment the target sequence can be from 9-15 nucleotides, 10-12 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides.
[0105] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
[0106] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, or cell or tissue or cell culture can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides. [0107] Complementary sequences within a LDA, e.g., within an iR A agent as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over a portion of one or both nucleotide sequences. When base-pairing is over the entire length of both sequences, such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., modulation of gene expression via a R Ai pathway. For the case of longer sequences, (>30nt), mismatches may be as many as 10, 20, 30 or more up to 25% of the molecule. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsR A comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter
oligonucleotide, may yet be referred to as "fully complementary" for the purposes described herein.
[0108] "Complementary" sequences, as used herein, may also include, or be formed entirely from, non- Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non- Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.
[0109] The terms "complementary," "fully complementary" and "substantially
complementary" herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA or siR A, or between the antisense strand of a LDA and a target sequence, as will be understood from the context of their use.
[0110] As used herein, a polynucleotide that is "substantially complementary to at least part of a IncRNA transcript refers to a polynucleotide that is substantially complementary to a contiguous portion of the IncRNA of interest (e.g., an IncRNA transcript or gene). For example, a polynucleotide is complementary to at least a part of IncRNA if the sequence is substantially complementary to a non- interrupted portion of a IncRNA transcript or gene.
[0111] "G," "C," "A," "T" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. The skilled person is also aware that a representation of an oligonucleotide as DNA may also be construed as RNA if the "T" nucleotides of the DNA are replaced in the sequence representation by "U".
[0112] In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target IncRNA. US Patent 7,732,593 describes constructs forming G-Uwobble base pairs and is incorporated herein by reference in its entirety. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.
[0113] The skilled artisan will recognize that the term "RNA molecule" or "ribonucleic acid molecule" encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a "ribonucleoside" includes a nucleoside base and a ribose sugar, and a "ribonucleotide" is a ribonucleoside with one, two or three phosphate moieties. However, the terms "ribonucleoside" and "ribonucleotide" can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below.
However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2'-0-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, 5' phosphate group, 5' triphosate group, 5' phosphorodithioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino-modified nucleoside, 2'-alkyl-modified nucleoside, 2'- alkoxyalkyl-modified nucleoside e.g., (2'-0-methoxy ethyl) nucleoside, morpholino nucleoside, an LNA nucleoside, a BNA nucleoside, a FHNA nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway or inhibit the function by steric effects such as translation arrest or modulation.
[0114] In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. However, it is self evident that under no circumstances is a double stranded DNA molecule encompassed by the term "iRNA."
[0115] In one aspect, an RNA interference agent (iRNA agent) includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Thus, in one aspect the invention relates to a single stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene, i.e., ssRNAi.
[0116] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a LDA, e.g., a dsRNA, siRNA or generally an iRNA agent. For example, when a 3 '-end of one strand of a dsRNA extends beyond the 5'- end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5' end , 3' end or both ends of either an antisense or sense strand of a dsRNA.
[0117] In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3 ' end and/or the 5' end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3 ' end and/or the 5' end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
[0118] In one embodiment, the sense strand of a dsRNA is connected with a biocleavable or biossable 1-25 nucleotide overhang at the 3 ' end and/or the 5' end capable of activating RNAse H. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleotide sequence functioning as an immunostimuatory agent or as an aptamer. In another embodiment, the 5 '-end of the sense strand or antisense stand or both strands carry a triphosphate capable of activating RIG-I protein. [0119] The terms "blunt" or "blunt ended" as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a "blunt ended" dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.
[0120] The term "antisense strand" or "guide strand" refers to the strand of an iR A agent, e.g., a dsRNA or siR A, which includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus. However, mismatches may be located in the internal positions of the molecule and on either strand of a dsRNA molecule.
[0121] The term "sense strand," or "passenger strand" as used herein, refers to the strand of a iRNA agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
Double-stranded ribonucleic acid (dsRNA)
[0122] The term "double-stranded RNA" or "dsRNA," as used herein, refers to an LDA that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having "sense" and "antisense" orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 50 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 50 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 46, 47, 48, 49 or 50 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self- complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a "hairpin loop") between the 3 '-end of one strand and the 5 '-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a "linker." The term "siRNA" while being an iRNA agent may also used herein to refer to a dsRNA as described above.
[0123] Described herein are IncRNA-directed agents (LDAs) that inhibit the expression of the IncRNA. In one embodiment, the LDA agent includes double-stranded ribonucleic acid
(dsRNA) molecules for inhibiting the expression of a IncRNA gene in a cell or mammal, e.g., in a human having a cancer or infectious disease, where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an RNA formed in the expression of a IncRNA, and where the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing the IncRNA, inhibits the expression of the IncRNA by at least 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method (to measure the proteins produced by any of the nearest neighbor genes or other protein coding gene known or believed to have a regulatory connection not a IncRNA), such as by Western blot. In one embodiment, the LDA agent activates the expression of a IncRNA in a cell or mammal. Expression of a IncRNA in cell culture, such as in COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells or in a biological sample from a subject can be assayed by measuring IncRNA RNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels of an associated protein coding gene (e.g., one indicative of IncRNA transcript levels), such as by immunofluorescence analysis, using, for example, Western Blotting or
flowcytometric techniques. [0124] A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an RNA formed during the expression of a IncRNA gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.
Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence.is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an IncRNA molecule. Where relevant, a "part" of an IncRNA target is a contiguous sequence of an IncRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some
circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
[0125] One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of 9 to 36, e.g., 15-30 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex of e.g., 15-30 base pairs that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. In another embodiment, a LDA agent useful to target IncRNA expression is generated in the target cell by cleavage of a larger dsRNA.
[0126] A dsRNA as described herein may further include one or more single-stranded nucleotide overhangs. The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are
commercially available from, for example, Biosearch, Applied Biosystems, Inc. In one embodiment, a IncRNA is a human IncRNA. In another embodiment the IncRNA is a mouse or a rat IncRNA. In yet another embodiment, the IncRNA is a cellular IncRNA to be targeted as a component or step in a bioprocessing reaction. [0127] In specific embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence referenced in Table 1, and the second sequence is selected from the group consisting of the corresponding antisense sequences of Table 1, the pairs of which are reported along with SEQ ID Nos of each sense:antisense pair. Pairs are listed with the sense strand first and then the antisense strand. For example, for the sense:antisense pair, (1,2), SEQ ID NO: 1 is the sense strand and SEQ ID NO: 2 is the antisense strand. Each IncRNA transcript (prefix "ENST") and IncRNA gene (prefix "ENSG") from which the dsRNA are designed are also disclosed in Table 1.
[0128] Alternative dsRNA agents that target elsewhere in the target sequence provided in Table 1 can readily be determined using the target sequence and the flanking IncRNA sequence.
[0129] In one aspect, a dsRNA will include one or more dsRNA nucleotide sequences, whereby the sense strand is selected from the groups of sequences provided in Table 1 the corresponding antisense strand of the sense strand selected from Table 1. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an RNA generated in the expression of a IncRNA. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Table 1, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand from Table 1. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.
[0130] The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et ah, EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 1 described herein can include at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter duplexes having one of the sequences of Table 1 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, or more contiguous nucleotides from one of the sequences of Table 1, and differing in their ability to inhibit the expression of a IncRNA by not more than 5, 10, 15, 20, 25, or 30 % inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.
[0131] In addition, the RNAs provided in Table 1 identify a site in a IncRNA transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of such sequences. As used herein, an iRNA agent is said to target within a particular site of an RNA transcript if the iRNA agent promotes cleavage of the transcript anywhere within that particular site. Such an iRNA agent will generally include at least 15 contiguous nucleotides from one of the sequences provided in Table 1 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a IncRNA. Given the identification of the sites for targeting provided by Table 1, it is also within the scope of the present invention for an LDA to target substantially the same location or site.
[0132] While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a "window" or "mask" of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence "window" progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Table 1 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively "walking the window" one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
[0133] Further, it is contemplated that for any sequence identified, e.g., in Table 1, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target IncRNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
[0134] An iRNA agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA agent as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA agent contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA agent contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5' or 3 ' end of the region of complementarity. For example, for a 23 nucleotide iRNA agent antisense strand which is complementary to a region of a IncRNA, the antisense strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA agent containing a mismatch to a target sequence is effective in inhibiting the expression of a IncRNA. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a IncRNA is important, especially if the particular region of complementarity in a IncRNA is known to have polymorphic sequence variation within the population.
[0135] In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. In yet another embodiment, the RNA of a LDA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.) 3 ' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.
[0136] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.
[0137] Representative U.S. patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5, 177, 195; 5, 188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321, 131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519, 126; 5,536,821; 5,541,316; 5,550, 11 1; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028, 188; 6,124,445; 6,160, 109; 6, 169, 170; 6, 172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683, 167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and US Pat RE39464, each of which is herein incorporated by reference.
[0138] Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
[0139] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5, 166,315; 5, 185,444; 5,214, 134; 5,216, 141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;
5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704;
- I l l - 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein
incorporated by reference.
[0140] In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
[0141] Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular— CH2— NH— CH2— , -CH2--N(CH3)--0--CH2- [known as a methylene (methylimino) or MMI backbone], ~CH2~0- -N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone is represented as— O— P— O— CH2— ] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
[0142] Modified RNAs may also contain one or more substituted sugar moieties. The LDAs, e.g., dsRNAs, featured herein can include one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include 0[(CH2)nO] mCH3, 0(CH2).nOCH3,
0(CH2)nNH2, 0(CH2) nCH3, 0(CH2)nONH2, and 0(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2' position: Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an iRNA agent, or a group for improving the pharmacodynamic properties of an iRNA agent, and other substituents having similar properties. In some embodiments, the modification includes a 2'-methoxyethoxy (2'-0— CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et ah, Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0-CH2-0-CH2-N(CH3)2, also described in examples herein below.
[0143] Other modifications include 2'-methoxy (2'-OCI¾), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the RNA of a LDA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl, cyclohexenyl (CeNA), Hexose (HNA), FHNA moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,1 18,800; 5,319,080; 5,359,044; 5,393,878; 5,446, 137; 5,466,786; 5,514,785;
5,519, 134; 5,567,81 1; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.
[0144] An iR A agent may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5 -hydroxy methyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3- deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et ah, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5 -substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar
modifications.
[0145] Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5, 134,066; 5,175,273; 5,367,066; 5,432,272; 5,457, 187; 5,459,255; 5,484,908; 5,502, 177; 5,525,71 1;
5,552,540; 5,587,469; 5,594, 121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6, 147,200;
6,166, 197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;
7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
[0146] The RNA of a LDA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo structural conformation. Also within the present invention are the use of bicyclic nucleic acids, carbocyclic LNAs and amino LNAs. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al, (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al, (2007) Mol Cane Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
[0147] Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084, 125; and 7,399,845, each of which is herein incorporated by reference in its entirety.
[0148] Another modification of the RNA of a LDA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the LDA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem. Let., 1994, 4: 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al, Biorg. Med. Chem. Let, 1993, 3 :2765-2770), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J, 1991, 10: 1 11 1-1 118; Kabanov et al, FEBS Lett, 1990, 259:327-330; Svinarchuk et al, Biochimie, 1993, 75:49-54), a phospholipid, e.g., di- hexadecyl-rac-glycerol or triethyl-ammonium l,2-di-0-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36:3651-3654; Shea et al, Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra ei a/., Biochim. Biophys. Acta, 1995, 1264:229- 237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923-937).
[0149] Disclosed in PCT/US2004/01 1829 filed 16-Apr-2004 (Applicant docket number ALN-018) and PCT/US2005/014472 filed 27-Apr-2005 (Applicant docket number ALN-026), each of which is incorporated herein in its entirey are conjugates of iRNA agents and certain moieties useful in this embodiment of the invention. Disclosed in PCT/US08/85577 filed 04- Dec-2008 and PCT/US08/85574 filed 04-Dec-2008 and PCT/US08/85582 filed 04-Dec-2008 (Applicant docket number ALN-047), each of which is incorporated herein in its entirey, are folate and other sugar conjugates useful in the present invention.
[0150] In one embodiment, a ligand alters the distribution, targeting or lifetime of a LDA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
[0151] Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L- lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, PBAVE polymers with or without CDM linkers or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide- polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
[0152] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectins, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl- galactos amine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. These ligands can be either monovalent or polyvalent.
[0153] Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross- linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g.
EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, lithocholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, Ibuprofen, Naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
[0154] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactos amine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.
[0155] The ligand can be a substance, e.g, a drug, which can increase the uptake of the LDA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
[0156] In one ligand, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
[0157] A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
[0158] In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non- kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
[0159] In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
[0160] In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).
[0161] In another aspect, the ligand is a cell-permeation agent, preferably a helical cell- permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase. [0162] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three- dimensional structure similar to a natural peptide. The attachment of peptide and
peptidomimetics to IncRNA-directed agents can affect pharmacokinetic distribution of the LDA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
[0163] A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS).
[0164] An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et ah, Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an LDA to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et ah, Cancer Gene Therapy 8:783-787, 2001).
Preferably, the RGD peptide will facilitate targeting of a LDA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing ayfi3 (Haubner et ah, Jour. Nucl. Med., 42:326-336, 2001).
[0165] Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313;
5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5, 109, 124; 5,1 18,802; 5, 138,045;
5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;
4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;
5,1 12,963; 5,214, 136; 5,082,830; 5,1 12,963; 5,214, 136; 5,245,022; 5,254,469; 5,258,506;
5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;
5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481 ; 5,587,371;
5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941 ; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which is herein incorporated by reference.
[0166] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within a LDA. The present invention also includes iRNA compounds that are chimeric compounds. "Chimeric" iRNA compounds or "chimeras," in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the LDA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the LDA may serve as a substrate for enzymes capable of cleaving
RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the IncRNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
[0167] In certain instances, the RNA of a LDA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the LDA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al, Biochem. Biophys. Res. Comm., 2007, 365(1):54-61 ; Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid
(Manoharan et al, Bioorg. Med. Chem. Lett., 1994, 4: 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al, Bioorg. Med. Chem. Let., 1993, 3 :2765), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J., 1991, 10: 11 1 ; Kabanov et al, FEBS Lett, 1990, 259:327; Svinarchuk et al, Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36:3651; Shea et al, Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate. iRNA agent structure
[0168] An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5 ' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
[0169] In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent will preferably have one or more of the following properties:
(1) it will be of the Formula 1, 2, 3, or 4 set out below;
(2) if single stranded it will have a 5 ' modification which includes one or more phosphate groups or one or more analogs of a phosphate group;
(3) it will, despite modifications, even to a very large number, or all of the nucleosides, have an antisense strand that can present bases (or modified bases) in the proper three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA;
(4) it will, despite modifications, even to a very large number, or all of the nucleosides, still have "RNA-like" properties, i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2' fluoro in place of 2' hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2' position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a Cy-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix.
Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures.
(Generally, it is preferred that a modified moiety at the 2' sugar position will be able to enter into H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a Cy-endo pucker in all, or at least 50, 75,80, 85, 90, or 95 % of its sugars; exhibit a Cy-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, orl sugar which is not a Cy-endo pucker structure. These limitations are particularly preferably in the antisense strand;
(5) regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70 % of the nucleotides in the molecule, or more than 50, 60, or 70 % of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2' position, are excluded from the definition of RNA agent.
[0170] A "single strand iRNA agent" as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents are preferably antisense with regard to the target molecule. In preferred embodiments single strand iRNA agents are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5'- phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5 '-monophosphate ((HO)2(0)P-0-5'); 5 '-diphosphate
((HO)2(0)P-0-P(HO)(0)-0-5'); 5'-triphosphate ((HO)2(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5'-(HO)(0)P-0-(HO)(0)P-0- P(HO)(0)-0-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-0-5'-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'-monothiophosphate
(phosphorothioate; (HO)2(S)P-0-5'); 5'-monodithiophosphate (phosphorodithioate;
(HO)(HS)(S)P-0-5'), 5'-phosphorothiolate ((HO)2(0)P-S-5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha- thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((ΗΟ)2(0)Ρ-ΝΗ-5', (ΗΟ)(ΝΗ2)(0)Ρ-0-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(0)-0-5'-, (OH)2(0)P-5'-CH2-), 5'-alkyletherphosphonates
(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(0)-0-5'-). (These modifications can also be used with the antisense strand of a double stranded iRNA.) If the iRNA agent is a single strand it is particularly preferred that it include a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
[0171] A single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target IncRNA. A single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50 nnucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.
[0172] Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3', and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length.
[0173] RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et ah, (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified "RNAs," they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of all of the above are discussed herein.
[0174] Much of the discussion below refers to single strand molecules. In many
embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described elsewhere herein.
[0175] As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and infact in most cases it will not. By way of example, a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5' end or ends can be phosphorylated.
[0176] In some embodiments it is particularly preferred, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5' or 3 ' overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3 ' or 5' overhang will be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2' OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence.
[0177] Modifications and nucleotide surrogates are discussed below.
Figure imgf000125_0001
FORMULA 1
[0178] The scaffold presented above in Formula 1 represents a portion of a ribonucleic acid. The basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers. Where the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine, the sugars are the unmodified 2' hydroxyl ribose sugar (as depicted) and W, X, Y, and Z are all O, Formula 1 represents a naturally occurring unmodified
oligoribonucleotide.
[0179] Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases.
Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Umodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent.
[0180] Modified nucleic acids and nucleotide surrogates can include one or more of:
(i) alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one of the positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid. However, for simplicity of terminology, except where otherwise noted, the W position at the 5' end of a nucleic acid and the terminal Z position at the 3 ' end of a nucleic acid, are within the term "linking phosphate oxygens" as used herein.);
(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose, e.g., as described herein;
(iii) wholesale replacement of the phosphate moiety (bracket I) with "dephospho" linkers;
(iv) modification or replacement of a naturally occurring base;
(v) replacement or modification of the ribose-phosphate backbone (bracket II);
(vi) modification of the 3' end or 5' end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g. a fluorescently labeled moiety, to either the 3' or 5' end of RNA.
[0181] The terms replacement, modification, alteration, and the like, as used in this context, do not imply any process limitation, e.g., modification does not mean that one must start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid bur rather modified simply indicates a difference from a naturally occurring molecule.
[0182] It is understood that the actual electronic structure of some chemical entities cannot be adequately represented by only one canonical form (i.e. Lewis structure). While not wishing to be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures. Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or "localization" of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as the predominant resonance form for a particular species. For example, any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen) would be represented by X = O and Y = N in the above figure.
[0183] Specific modifications are discussed in more detail below. The Phosphate Group
[0184] The phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i.e., X and Y in Formula 1 above). However, the phosphate group can be modified by replacing one of the oxygens with a different substituent. One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
[0185] Examples of modified phosphate groups include phosphorothioate,
phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroami dates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which the individual diastereomers exhibit varying resistance to nucleases.
Further, the hybridization affinity of RNA containing chiral phosphate groups can be lower relative to the corresponding unmodified RNA species. Thus, while not wishing to be bound by theory, modifications to both X and Y which eliminate the chiral center, e.g. phosphorodithioate formation, may be desirable in that they cannot produce diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred.
[0186] The phosphate linker can also be modified by replacement of a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged
phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen (position W (3') or position Z (5'). Replacement of W with carbon or Z with nitrogen is preferred.
The Sugar Group
[0187] A modified RNA can include modification of all or some of the sugar groups of the ribonucleic acid. E.g., the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. While not being bound by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2' alkoxide ion. The 2' alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Again, while not wishing to be bound by theory, it can be desirable to some embodiments to introduce alterations in which alkoxide formation at the 2' position is not possible.
[0188] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), 0(CH2CH20)nCH2CH2OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; 0-ΑΜΓΝΕ (AMINE = N¾; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, 0(CH2)nAMINE, (e.g., AMINE = NI¾; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that
oligonucleotides containing only the methoxyethyl group (MOE), (OCH2CH2OCH3, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.
[0189] "Deoxy" modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. H2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMrNE (AMINE = NH2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), -NHC(0)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Preferred substitutents are 2'- methoxyethyl, 2'-OCH3, 2'-0-allyl, 2'-C- allyl, and 2'-fluoro.
[0190] The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar.
[0191] Modified RNA's can also include "abasic" sugars, which lack a nucleobase at C-Γ. These abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms.
[0192] To maximize nuclease resistance, the 2' modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called "chimeric" oligonucleotides are those that contain two or more different modifications.
[0193] The modificaton can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs.
Replacement of the Phosphate Group
[0194] The phosphate group can be replaced by non-phosphorus containing connectors (cf. Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety. [0195] Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methylenecarbonylamino and methylenemethylimino groups.
Replacement of Ribophosphate Backbone
[0196] Oligonucleotide- mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone.
[0197] Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.
Terminal Modifications
[0198] The 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end, 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. E.g., the 3' and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer. The terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3 ' or C-5' O, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., - (CH2)n-, -(CH2)nN-, -(CH2)nO-, -(CH2)nS-, 0(CH2CH20)nCH2CH2OH (e.g., n = 3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. When a spacer/phosphate-functional molecular entity-spacer/phosphate array is interposed between two strands of iRNA agents, this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3 ' end can be an - OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).
[0199] Terminal modifications can be added for a number of reasons, including as discussed elsewhere herein to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or phosphate analogs. E.g., in preferred embodiments iR A agents, especially antisense strands, are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5 '-monophosphate ((HO)2(0)P-0-5'); 5 '-diphosphate ((HO)2(0)P-0- P(HO)(0)-0-5'); 5 '-triphosphate ((HO)2(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5'-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'- adenosine cap (Appp), and any modified or unmodified nucleotide cap structure ( -O-5'- (HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'); 5'-monothiophosphate (phosphorothioate;
(HO)2(S)P-0-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-0-5'), 5'- phosphorothiolate ((HO)2(0)P-S-5'); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma- thiotriphosphate, etc.), 5'-phosphoramidates ((ΗΟ)2(0)Ρ-ΝΗ-5', (ΗΟ)(ΝΗ2)(0)Ρ-0-5'), 5'- alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(0)-0-5'-, (OH)2(0)P-5'-CH2-), 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(0)-0-5'-).
[0200] Terminal modifications useful for increasing resistance to degradation include
[0201] Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross- linking an RNA agent to another moiety; modifications useful for this include mitomycin C.
The Bases
[0202] Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications. Alternatively, substituted or modified analogs of any of the above bases, e.g., "unusual bases" and "universal bases," can be employed. Examples include without limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5- azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2- thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3- methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6- isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613.
[0203] Generally, base changes are less preferred for promoting stability, but they can be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent. Modified bases can reduce target specificity. This should be taken into consideration in the design of iRNA agents.
Evaluation of Candidate RNA's [0204] One can evaluate a candidate RNA agent, e.g., a modified RNA, for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property. For example, resistance to a degradent can be evaluated as follows. A candidate modified RNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu, which includes a degradative agent, e.g., a nuclease. E.g., one can use a biological sample, e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells. The candidate and control could then be evaluated for resistance to degradation by any of a number of approaches. For example, the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule.
[0205] A functional assay can also be used to evaluate the candidate agent. A functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence lncRNA expression. For example, a cell, e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added. Efficacy of the candidate agent on gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents.
References
General References
[0206] The oligoribonucleotides and oligoribonucleosides used in accordance with this invention may be with solid phase synthesis, see for example "Oligonucleotide synthesis, a practical approach", Ed. M. J. Gait, IRL Press, 1984; "Oligonucleotides and Analogues, A Practical Approach", Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine- aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2'-0— Methyloligoribonucleotide- s: synthesis and applications, Chapter 4,
Phosphorothioate oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2'-deoxyribonucleoside methylphosphonates, and. Chapter 7, Oligodeoxynucleotides containing modified bases. Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223- 2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references referred to therein.
[0207] Modification described in WO 00/44895, WOO 1/75164, or WO02/44321 can be used herein.
Phosphate Group References
[0208] The preparation of phosphinate oligoribonucleotides is described in U.S. Pat. No. 5,508,270. The preparation of alkyl phosphonate oligoribonucleotides is described in U.S. Pat. No. 4,469,863. The preparation of phosphoramidite oligoribonucleotides is described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation of phosphotriester
oligoribonucleotides is described in U.S. Pat. No. 5,023,243. The preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5, 130,302 and 5, 177, 198. The preparation of 3'-Deoxy-3'-amino phosphoramidate oligoribonucleotides is described in U.S. Pat. No. 5,476,925. 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801. Preparation of sulfur bridged nucleotides is described in Sproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. Tetrahedron Lett. 1989, 30, 4693.
Sugar Group References
[0209] Modifications to the 2' modifications can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. Specific modifications to the ribose can be found in the following references: 2'-fluoro (Kawasaki et. al, J. Med. Chem., 1993, 36, 831 - 841), 2'-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938), "LNA" (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).
Replacement of the Phosphate Group References
[0210] Methylenemethylimino linked oligoribonucleosides, also identified herein as MMI linked oligoribonucleosides, methylenedimethylhydrazo linked oligoribonucleosides, also identified herein as MDH linked oligoribonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified herein as amide-3 linked oligoribonucleosides, and
methyleneaminocarbonyl linked oligonucleosides, also identified herein as amide-4 linked oligoribonucleosides as well as mixed backbone compounds having, as for instance, alternating MMI and PO or PS linkages can be prepared as is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and in published PCT applications PCT/US92/04294 and
PCT/US92/04305 (published as WO 92/20822 WO and 92/20823, respectively). Formacetal and thioformacetal linked oligoribonucleosides can be prepared as is described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linked oligoribonucleosides can be prepared as is described in U.S. Pat. No. 5,223,618. Siloxane replacements are described in Cormier,J.F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements are described in Tittensor, J.R. J. Chem. Soc. C 1971, 1933. Carboxymethyl replacements are described in Edge, M.D. et al. J. Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are described in Stirchak, E.P. Nucleic Acids Res. 1989, 17, 6129.
Replacement of the Phosphate-Ribose Backbone References
[0211] Cyclobutyl sugar surrogate compounds can be prepared as is described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared as is described in U.S. Pat. No. 5,519, 134. Morpholino sugar surrogates can be prepared as is described in U.S. Pat. Nos.
5, 142,047 and 5,235,033, and other related patent disclosures. Peptide Nucleic Acids (PNAs) are known per se and can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. No. 5,539,083.
Terminal Modification References
[0212] Terminal modifications are described in Manoharan, M. et al. Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and references therein.
Bases References
[0213] N-2 substitued purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,457, 191. 5,6-Substituted pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,484,908. Additional references can be disclosed in the above section on base modifications. SHEET INTENTIONALLY LEFT BLANK
Preferred iRNA Agents
[0214] Preferred RNA agents have the following structure (see Formula 2 below):
Figure imgf000136_0001
FORMULA 2
[0215] Referring to Formula 2 above, R1, R2, and R3 are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5- azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7- deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3- methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5 -nitro indole, 3-nitropyrrole, 5- methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino- 3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N- methylguanines, or O-alkylated bases.
[0216] R4, R5, and R6 are each, independently, OR8, 0(CH2CH20)mCH2CH2OR8; 0(CH2)nR9; 0(CH2)nOR9, H; halo; NH2; NHR8; N(R8)2; NH(CH2CH2NH)mCH2CH2NHR9; NHC(0)R8; ; cyano; mercapto, SR8; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl,
alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or R4, R5, or R6 together combine with R7 to form an [-0-CH2-] covalently bound bridge between the sugar 2' and 4' carbons.
[0217] A1 is:
Figure imgf000138_0001
; H; OH; OCH3; W1; an abasic nucleotide; or absent;
(a preferred Al , especially with regard to anti-sense strands, is chosen from 5'- monophosphate ((ΗΟ)2(0)Ρ-0-5'), 5'-diphosphate ((ΗΟ)2(0)Ρ-0-Ρ(ΗΟ)(0)-0-5'), 5'- triphosphate ((ΗΟ)2(0)Ρ-0-(ΗΟ)(0)Ρ-0-Ρ(ΗΟ)(0)-0-5'), 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5'-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'), 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-0-5'-(HO)(0)P-0- (ΗΟ)(0)Ρ-0-Ρ(ΗΟ)(0)-0-5'), 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-0-5'), 5'- monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-0-5'), 5'-phosphorothiolate
((HO)2(0)P-S-5'); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((ΗΟ)2(0)Ρ-ΝΗ-5', (ΗΟ)( Η2)(0)Ρ-0-5'), 5'-alkylphosphonates
(R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(0)-0-5'-, (OH)2(0)P-5'-CH2-), 5'- alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(0)-0-5'-)). [0218] A2 is:
Figure imgf000139_0001
[0219] A is:
Figure imgf000139_0002
; and
[0220] A4 is:
Figure imgf000140_0001
; H; Z4; an inverted nucleotide; an abasic nucleotide; or absent.
[0221] W1 is OH, (CH2)nR10, (CH2)nNHR10, (CH2)n OR10, (CH2)n SR10; 0(CH2)nR10;
0(CH2)nOR10, 0(CH2)nNR10, 0(CH2)nSR10; 0(CH2)nSS(CH2)nOR10, 0(CH2)nC(0)OR10, NH(CH2)nR10; NH(CH2)nNR10 ;NH(CH2)nOR10, NH(CH2)nSR10; S(CH2)nR10, S(CH2)nNR10, S(CH2)nOR10, S(CH2)nSR10 0(CH2CH20)mCH2CH2OR10; 0(CH2CH20)mCH2CH2NHR10 , NH(CH2CH2NH)mCH2CH2NHR10; Q-R10, O-Q-R10 N-Q-R10, S-Q-R10 or -0-. W4 is O, CH2, NH, or S.
[0222] X1, X2, X3, and X4 are each, independently, O or S.
[0223] Y1, Y2, Y3, and Y4 are each, independently, OH, O", OR8, S, Se, BH3 ", H, NHR9, N(R9)2 alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.
[0224] Z1, Z2, and Z3 are each independently O, CH2, NH, or S. Z4 is OH, (CH2)nR10, (CH2)nNHR10, (CH2)n OR10, (CH2)n SR10; 0(CH2)nR10; 0(CH2)nOR10, 0(CH2)nNR10,
0(CH2)nSR10, 0(CH2)nSS(CH2)nOR10, 0(CH2)nC(0)OR10; NH(CH2)nR10; NH(CH2)nNR10 ;NH(CH2)nOR10, NH(CH2)nSR10; S(CH2)nR10, S(CH2)nNR10, S(CH2)nOR10, S(CH2)nSR10 0(CH2CH20)mCH2CH2OR10, 0(CH2CH20)mCH2CH2NHR10 ,
NH(CH2CH2NH)mCH2CH2NHR10; Q-R10, O-Q-R10 N-Q-R10, S-Q-R10.
[0225] x is 5-100, chosen to comply with a length for an RNA agent described herein.
[0226] R7 is H; or is together combined with R4, R5, or R6 to form an [-0-CH2-] covalently bound bridge between the sugar 2' and 4' carbons.
[0227] R8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R9 is NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and R is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid,myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles); or an RNA agent, m is 0-1,000,000, and n is 0-20. Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents.
[0228] Preferred iRNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):
Figure imgf000142_0001
FORMULA 3
[0229] Referring to Formula 3, A10-A40 is L-G-L; A10 and/or A40 may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH2(CH2)g; N(CH2)g; 0(CH2)g; S(CH2)g. G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and
methyleneoxymethylimino.
[0230] R10, R20, and R30 are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5- halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7- alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6- diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone, 5 -nitro indole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5- methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5- methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3 -methylcytosine, 5- methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2- methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.
[0231] R40, R50, and R60 are each, independently, OR8, 0(CH2CH20)mCH2CH2OR8;
0(CH2)nR9; 0(CH2)nOR9, H; halo; NH2; NHR8; N(R8)2; NH(CH2CH2NH)mCH2CH2R9;
NHC(0)R8;; cyano; mercapto, SR7; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups; or R40, R50, or R60 together combine with R70 to form an [-0- CH2-] covalently bound bridge between the sugar 2' and 4' carbons.
[0232] x is 5-100 or chosen to comply with a length for an RNA agent described herein.
[0233] R™ is H; or is together combined with R40, R50, or R60 to form an [-0-CH2-] covalently bound bridge between the sugar 2' and 4' carbons.
[0234] R8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and R9 is NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid, m is 0-1,000,000, n is 0-20, and g is 0-2.
[0235] Preferred nucleoside surrogates have the following structure (see Formula 4 below):
SLR100-(M-SLR200)X-M-SLR300
FORMULA 4
[0236] S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CH2(CH2)g; N(CH2)g; 0(CH2)g; S(CH2)g; -C(0)(CH2)n-or may be absent. M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent. [0237] R , R , and R are each, independently, H (i.e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5- halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7- alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6- diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1, 2, 4,-triazoles, 2- pyridinones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5- methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5- methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5- methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2- methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases,
x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2.
Nuclease resistant monomers
[0238] In one aspect, the invention features a nuclease resistant monomer, or a an iRNA agent which incorporates a nuclease resistant monomer (NMR), such as those described herein and those described in copending, co-owned United States Provisional Application Serial No.
60/469,612 (Attorney Docket No. 14174-069P01), filed on May 9, 2003, which is hereby incorporated by reference.
[0239] In addition, the invention includes iRNA agents having a NMR and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a NMR.
[0240] An iRNA agent can include monomers which have been modifed so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRM's, or nuclease resistance promoting monomers or modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC (RNA-induced Silencing Complex), or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.
[0241] While not wishing to be bound by theory, it is believed that modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex. Preferred modifications are those that increase exonuclease and endonuclease resistance and thus prolong the halflife of the iRNA agent prior to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex. Again, while not wishing to be bound by any theory, it is believed that placement of the modifications at or near the 3 ' and/or 5' end of antisense strands can result in iRNA agents that meet the preferred nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that placement of the modifications at e.g., the middle of a sense strand can result in iRNA agents that are relatively less likely to undergo off-targeting.
[0242] Modifications described herein can be incorporated into any double-standed RNA and RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the modifications described herein. The anti sense strand may include modifications at the 3' end and/or the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sense strand may include modifications at the 3 ' end and/or the 5' end and/or at any one of the intervening positions between the two ends of the strand. The iRNA agent may also include a duplex comprising two hybridized antisense strands. The first and/or the second antisense strand may include one or more of the modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3' end and/or the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular configurations are discussed below.
[0243] Modifications that can be useful for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone:
(i) chiral (Sp) thioates. Thus, preferred NRM's include nucleotide dimers with an enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., Sp or Rp, at the position X, where this is the position normally occupied by the oxygen. The atom at X can also be S, Se, Nr2, or Br3. When X is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Such NRM's are discussed in more detail below;
(ii) attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. Thus, preferred NRM's include monomers at the terminal position derivitized at a cationic group. As the 5' end of an antisense sequence should have a terminal -OH or phosphate group this NRM is preferraly not used at th 5' end of an anti-sense sequence. The group should be attached at a position on the base which minimizes intererence with H bond formation and hybridization, e.g., away form the face which intereacts with the complementary base on the other strand, e.g, at the 5' position of a pyrimidine or a 7-position of a purine. These are discussed in more detail below;
(iii) nonphosphate linkages at the termini. Thus, preferred NRM's include Non- phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a phosphate bond. Examples include 3' CH2-NCH3-0-CH2-5' and 3' CH2-NH-(0=)-CH2- 5'.;
(iv) 3 '-bridging thiophosphates and 5'-bridging thiophosphates. Thus, preferred NRM's can included these structures;
(v) L-RNA, 2'-5' likages, inverted linkages, a-nucleosides. Thus, other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2'-5' phosphate, non-phosphate and modified phosphate linkages (e.g., thiophospahtes,
phosphoramidates and boronophosphates); dimers having inverted linkages, e.g., 3 '-3' or 5'-5' linkages; monomers having an alpha linkage at the site on the sugar, e.g., the structures described herein having an alpha linkage;
(vi) conjugate groups. Thus, preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar , base, or backbone ;
(vi) abasic linkages. Thus, preferred NRM's can include an abasic monomer, e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein.; and
(vii) 5'-phosphonates and 5'-phosphate prodrugs. Thus, preferred NRM's include monomers, preferably at the terminal position, e.g., the 5' position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result of the action of a component in the subject's body, e.g, a carboxyesterase or an enzyme present in the subject's body. E.g., a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which attacks a carbon adjacent to the O of a phosphate and resulting in the production of an uprotected phosphate.
[0244] One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintainted.
[0245] In some embodiments NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off-target silencing.
Chiral Sp Thioates
[0246] A modification can include the alteration, e.g., replacement, of one or both of the non- linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar surrogate-base moities, SBi and SB2.
Figure imgf000147_0001
FORMULA X
[0247] In certain embodiments, one of the non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR2 (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non-linking oxygens with one of the above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphorus atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). Thus if 60% of a population of stereogenic phosphorus atoms have the Rp configuration, then the remaining 40% of the population of stereogenic phosphorus atoms have the Sp configuration.
[0248] In some embodiments, iRNA agents, having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the Sp configuration. Alternatively, iRNA agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the Rp configuration. In other embodiments, the population of stereogenic phosphorus atoms may have the Sp configuration and may be substantially free of stereogenic phosphorus atoms having the Rp configuration. In still other embodiments, the population of stereogenic phosphorus atoms may have the Rp configuration and may be substantially free of stereogenic phosphorus atoms having the Sp configuration. As used herein, the phrase
"substantially free of stereogenic phosphorus atoms having the Rp configuration" means that moieties containing stereogenic phosphorus atoms having the Rp configuration cannot be detected by conventional methods known in the art (chiral HPLC, lH NMR analysis using chiral shift reagents, etc.). As used herein, the phrase "substantially free of stereogenic phosphorus atoms having the Sp configuration" means that moieties containing stereogenic phosphorus atoms having the Sp configuration cannot be detected by conventional methods known in the art (chiral HPLC, XH NMR analysis using chiral shift reagents, etc.).
[0249] In a preferred embodiment, modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom. In an especially preferred embodiment, the population of phosphorothioate stereogenic phosphorus atoms may have the Sp configuration and be substantially free of stereogenic phosphorus atoms having the RP configuration. [0250] Phosphorothioates may be incorporated into iR A agents using dimers e.g., formulas X-l and X-2. The former can be used to introduce phosphorothioate
Figure imgf000149_0001
X-l X-2
at the 3 ' end of a strand, while the latter can be used to introduce this modification at the 5' end or at a position that occurs e.g., 1, 2, 3, 4, 5, or 6 nucleotides from either end of the strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, !¾' can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH3), DMT is dimethoxytrityl, and "BASE" can be a natural, unusual, or a universal base.
[0251] X-l and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms having essentially only the Rp configuration (i.e., being substantially free of the Sp configuration) or only the Sp configuration (i.e., being substantially free of the Rp configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphorus atoms in which about 50% of the atoms have the Rp configuration and about 50% of the atoms have the Sp
configuration. Dimers having stereogenic phosphorus atoms with the Rp configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the Sp configuration using e.g., enzymatic degradation and/or conventional chromatography techniques. Cationic Groups
[0252] Modifications can also include attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. A cationic group can be attached to any atom capable of substitution on a natural, unusual or universal base. A preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. A cationic group can be attached e.g., through the C2' position of a sugar or analogous position in a cyclic or acyclic sugar surrogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., 0(CH2)nAMINE, (e.g., AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or NH(CH2CH2NH)nCH2CH2-AMrNE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino).
Nonphosphate Linkages
[0253] Modifications can also include the incorporation of nonphosphate linkages at the 5' and/or 3' end of a strand. Examples of nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo,
methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methyl phosphonate and hydroxylamino groups.
[0254] 3 '-bridging thiophosphates and 5 '-bridging thiophosphates; iocked-RNA, 2 '-5 ' likages, inverted linkages, a-nucleosides; conjugate groups; abasia linkages; and 5 '- phosphonates and 5 '-phosphate prodrugs
[0255] Referring to formula X above, modifications can include replacement of one of the bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom..
[0256] Modifications can also include linking two sugars via a phosphate or modified phosphate group through the 2' position of a first sugar and the 5' position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are eached linked through the respective3' positions. Modified RNA's can also include "abasic" sugars, which lack a nucleobase at C- . The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar. In another subset of this modification, the natural, unusual, or universal base may have the a-configuration. Modifcations can also include L-RNA.
[0257] Modifications can also include 5'-phosphonates, e.g., P(0)(0")2-X-C5 -sugar (X= CH2, CF2, CHF and 5'-phosphate prodrugs, e.g., P(0)[OCH2CH2SC(0)R]2CH2C5'-sugar. In the latter case, the prodrug groups may be decomposed via reaction first with carboxy esterases. The remaining ethyl thiolate group via intramolecular SN2 displacement can depart as episulfide to afford the underivatized phosphate group.
[0258] Modification can also include the addition of conjugating groups described elseqhere herein, which are prefereably attached to an iRNA agent through any amino group available for conjugation.
[0259] Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit hybridization so it is preferably to use them only in terminal regions, and preferrable to not use them at the cleavage site or in the cleavage region of an sequence which targets a subject sequence or gene.. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some
embodiments it is desirabable to put the NRM at the cleavage site or in the cleavage region of a sequence which does not target a subject sequence or gene, as it can minimize off-target silencing.
[0260] In addition, an iRNA agent described herein can have an overhang which does not form a duplex structure with the other sequence of the iRNA agent— it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence of the iRNA agent.
[0261] In most cases, the nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which interfer with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et ah, 2001, Genes and Dev. 15: 188, hereby incorporated by reference, cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 1 1 nucleotides upstream of the first nucleotide which is complementary to the guide sequence. As used herein cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means an nucleotide with 1, 2, or 3 nucletides of the cleave site, in either direction.)
[0262] Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject.
[0263] An iRNA agent can have a first and a second strand chosen from the following:
a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;
a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region;
a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3 ' and the 5' end; and
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;
a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3 ' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand).
[0264] An iRNA agent can also target two sequences and can have a first and second strand chosen from:
a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;
a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);
a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
a first strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;
a first strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand) and a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;
a second strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3 ' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand).
Ribose Mimics
[0265] In one aspect, the invention features a ribose mimic, or an iRNA agent which incorporates a ribose mimic, such as those described herein and those described in copending co- owned United States Provisional Application Serial No. 60/454,962 (Attorney Docket No.
14174-064P01), filed on March 13, 2003, which is hereby incorporated by reference.
[0266] In addition, the invention includes iRNA agents having a ribose mimic and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a ribose mimic.
[0267] Thus, an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent. Nucleases, e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid. The secondary hydroxy group confers nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the modification. While not wishing to be bound by theory, it is believed that the presence of a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3 ' ribose hydroxyl group, thereby causing it to be less susceptible to degradation.
[0268] The secondary hydroxyl group refers to an "OH" radical that is attached to a carbon atom substituted by two other carbons and a hydrogen. The secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group. The hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base.. The hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group.
[0269] Preferred iRNA agent delivery modules with five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group have the following structure (see formula Y below):
Figure imgf000155_0001
00
[0270] Referring to formula Y, A is an iRNA agent, including any iRNA agent described herein. The iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to "W" of the phosphate group. These spacers or linkers can include e.g., -(CH2)n-, -(CH2)nN-, - (CH2)nO-, -(CH2)nS-, 0(CH2CH20)nCH2CH2OH (e.g., n = 3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents.
[0271] The iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group, W and Z can be independently NH, O, or S; and X and Y can be independently S, Se, BH3 ", C\-Ce alkyl, C6-C10 aryl, H, O, O", alkoxy or amino (including alkylamino, arylamino, etc.). Preferably, W, X and Z are O and Y is
S. [0272] Ri and R3 are each, independently, hydrogen; or C1-C100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.
[0273] R2 is hydrogen; C1-C100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R2 may be taken together with with R4 or R^ to form a ring of 5-12 atoms.
[0274] R4 is hydrogen; C1-C100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R4 may be taken together with with R2 or R5 to form a ring of 5-12 atoms.
[0275] R5 is hydrogen, C1-C100 alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R5 may be taken together with with R4 to form a ring of 5-12 atoms.
[0276] R6 is hydrogen, C1-C100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when n is 1, R6 may be taken together with with R2 to form a ring of 6-10 atoms;
[0277] R7 is hydrogen, C1-C100 alkyl, or C(0)(CH2)qC(0)NHR9; T is hydrogen or a functional group; n and q are each independently 1-100; Rs is C1-C10 alkyl or C6-C10 aryl; and R9 is hydrogen, CI -CIO alkyl, C6-C10 aryl or a solid support agent.
[0278] Preferred embodiments may include one of more of the following subsets of iRNA agent delivery modules.
[0279] In one subset of RNAi agent delivery modules, A can be connected directly or indirectly through a terminal 3 ' or 5 ' ribose sugar carbon of the RNA agent.
[0280] In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S.
[0281] In still yet another subset of RNAi agent delivery modules, n is 1, and R2 and R6 are taken together to form a ring containing six atoms and R4 and R5 are taken together to form a ring containing six atoms. Preferably, the ring system is a trans-dscal . For example, the RNAi agent delivery module of this subset can include a compound of Formula (Y-l):
Figure imgf000156_0001
[0282] The functional group can be, for example, a targeting group (e.g., a steroid or a carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety). The targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin.
[0283] As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art.The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization.
Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
Ribose Replacement Monomer Subunits
[0284] iRNA agents can be modified in a number of ways which can optimize one or more characteristics of the iRNA agent. In one aspect, the invention features a ribose replacement monomer subunit (RRMS), or a an iRNA agent which incorporates a RRMS, such as those described herein and those described in one or more of United States Provisional Application Serial No. 60/493,986 (Attorney Docket No. 14174-079P01), filed on August 8, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/494,597 (Attorney Docket No. 14174-080P01), filed on August 11, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/506,341 (Attorney Docket No. 14174-080P02), filed on September 26, 2003, which is hereby incorporated by reference; and in United States Provisional Application Serial No. 60/158,453 (Attorney Docket No. 14174- 080P03), filed on November 7, 2003, which is hereby incorporated by reference.
[0285] In addition, the invention includes iRNA agents having a RRMS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an archtecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a RRMS.
[0286] The ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. A
ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.
[0287] The carriers further include (i) at least two "backbone attachment points" and (ii) at least one "tethering attachment point." A "backbone attachment point" as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A "tethering attachment point" as used herein refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property, e.g., lipophilicity, of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.
[0288] Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance.
Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target IncRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).
[0289] Accordingly, in one aspect, the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (R- 1) is incorporated into at least one of said strands.
Figure imgf000159_0001
(R-l)
[0290] Referring to formula (R- 1), X is N(CO)R7, NR7 or CH2; Y is NR8, O, S, CR9R10, or absent; and Z is CRUR12 or absent.
[0291] Each of R1, R2, R3, R4, R9, and R10 is, independently, H, ORa, ORb, (CH2)nORa, or (CH2)nORb, provided that at least one of R1, R2, R3, R4, R9, and R10 is ORa or ORb and that at least one of R1, R2, R3, R4, R9, and R10 is (CH2)nORa, or (CH2)nORb (when the RRMS is terminal, one of R1, R2, R3, R4, R9, and R10 will include Ra and one will include Rb; when the RRMS is internal, two of R1, R2, R3, R4, R9, and R10 will each include an Rb); further provided that preferably ORa may only be present with (CH2)nORb and (CH2)nORa may only be present with ORb.
[0292] Each of R5, R6, R11, and R12 is, independently, H, Ci-C6 alkyl optionally substituted with 1-3 R13, or C(0)NHR7; or R5 and R11 together are C3-C8 cycloalkyl optionally substituted with R14.
[0293] R7 is Ci-C20 alkyl substituted with NRcRd; R8 is Ci-C6 alkyl; R13 is hydroxy, d-C4 alkoxy, or halo; and R14 is NRCR7.
[0294] Ra is:
A
p B
C
; and Rb is:
-O Strand
[0295] Each of A and C is, independently, O or S.
[0296] B is OH, O", or
O O
O- -o- -OH
O" O"
[0297] Rc is H or C1-C6 alkyl; Rd is H or a ligand; and n is 1-4.
[0298] In a preferred embodiment the ribose is replaced with a pyrroline scaffold, and X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent.
[0299] In other preferred embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R7 or NR7, Y is CR9R10, and Z is CRnR12.
[0300] In other preferred embodiments the ribose is replaced with a piperazine scaffold, and X is N(CO)R7 or NR7, Y is NR8, and Z is CRnR12.
[0301] In other preferred embodiments the ribose is replaced with a morpholino scaffold, and X is N(CO)R7 or NR7, Y is O, and Z is CRnR12 .
[0302] In other preferred embodiments the ribose is replaced with a decalin scaffold, and X isCH2; Y is CR9R10; and Z is CRUR12; and R5 and R11 together are C6 cycloalkyl.
[0303] In other preferred embodiments the ribose is replaced with a decalin/indane scafold and , and X is CH2; Y is CR9R10; and Z is CRnR12; and R5 and R11 together are C5 cycloalkyl.
[0304] In other preferred embodiments, the ribose is replaced with a hydroxyproline scaffold.
[0305] RRMSs described herein may be incorporated into any double-stranded RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the RRMSs described herein. An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed at or near (within 1, 2, or 3 positions) of the 3 ' or 5' end of the sense strand or at near (within 2 or 3 positions of) the 3' end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end of the antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.
[0306] In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3 ' end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and at (or within 1, 2, or 3 positions of) the 3 ' end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both ligands are located at the same end of the iRNA agent.
[0307] In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.
[0308] In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3 ' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a "hairpin ligand."
[0309] In other embodiments, an iRNA agent may have an RRMS at the 3 ' end of the sense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.
[0310] In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. [0311] It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides. Most embodiments will have a 3 ' overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3' overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5' ends are preferably phosphorylated.
[0312] Preferred carriers have the general formula (R-3) provided below. (In that structure preferred backbone attachment points can be chosen from R1 or R2; R3 or R4; or R9 and R10 if Y is CR9R10 (two positions are chosen to give two backbone attachment points, e.g., R1 and R4, or R4 and R9. Preferred tethering attachment points include R7; R5 or R6 when X is CH2. The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R1 or R2; R3 or R4; or R9 or R10 (when Y is CR9R10), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be - CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.)
Figure imgf000162_0001
(R-3)
[0313] X is N(CO)R7, NR7 or CH2; Y is NR8, O, S, CR9R10; and Z is CRnR12 or absent.
[0314] Each of R1, R2, R3, R4, R9, and R10 is, independently, H, ORa, or (CH2)nORb, provided that at least two of R1, R2, R3, R4, R9, and R10 are ORa and/or (CH2)nORb.
[0315] Each of R5, R6, R11, and R12 is, independently, a ligand, H, Ci-C6 alkyl optionally substituted with 1-3 R13, or C(0)NHR7; or R5 and R11 together are C3-C8 cycloalkyl optionally substituted with R14. [0316] R7 is H, a ligand, or Ci-C20 alkyl substituted with NRcRd; R8 is H or Ci-C6 alkyl; R13 is hydroxy, C1-C4 alkoxy, or halo; R14 is NRCR7; R15 is Ci-Ce alkyl optionally substituted with cyano, or C2-C6 alkenyl; R16 is C1-C10 alkyl; and R17 is a liquid or solid phase support reagent.
[0317] L is -C(0)(CH2)qC(0)-, or -C(0)(CH2)qS-; Ra is CAr3; Rb is P(0)(0")H,
P(OR15)N(R16)2 or L-R17; Rc is H or Ci-C6 alkyl; and Rd is H or a ligand.
[0318] Each Ar is, independently, C6-C10 aryl optionally substituted with C1-C4 alkoxy; n is 1-4; and q is 0-4.
[0319] Exemplary carriers include those in which, e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent; or X is N(CO)R7 or NR7, Y is CR9R10, and Z is CRnR12; or X is N(CO)R7 or NR7, Y is NR8, and Z is CRnR12; or X is N(CO)R7 or NR7, Y is O, and Z is CRUR12; or X is CH2; Y is CR9R10; Z is CRnR12, and R5 and R11 together form C6 cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH2; Y is CR9R10; Z is CRUR12, and R5 and R11 together form C5 cycloalkyl (H, z = 1).
[0320] In certain embodiments, the carrier may be based on the pyrroline ring system or the 3-hydroxyproline ring system, e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent (D). OFG1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene
Figure imgf000163_0001
D group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (- CH2OFG1 in D). OFG2 is preferably attached directly to one of the carbons in the five- membered ring (-OFG2 in D). For the pyrroline-based carriers, -CH2OFG1 may be attached to C- 2 and OFG2 may be attached to C-3; or -CH2OFG1 may be attached to C-3 and OFG2 may be attached to C-4. . In certain embodiments, CH2OFG1 and OFG2 may be geminally substituted to one of the above-referenced carbons. For the 3-hydroxyproline-based carriers, -CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-4. The pyrroline- and 3-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CHzOFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.
[0321] In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is CRUR12. OFG1 is preferably
Figure imgf000164_0001
E attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=l) or ethylene group (n=2), connected to one of the carbons in the six-membered ring
Figure imgf000164_0002
in E]. OFG2 is preferably attached directly to one of the carbons in the six-membered ring (-OFG2 in E). -(CH^nOFG1 and OFG2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, - (CH2)nOFG1 and OFG2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH^nOFG1 may be attached to C-2 and OFG2 may be attached to C-3; -(CH^nOFG1 may be attached to C-3 and OFG2 may be attached to C-2; -(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-4; or -(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C-3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.
[0322] In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R7 or NR7, Y is NR8, and Z is CRUR12, or the morpholine ring system (G), e.g., X is N(CO)R7 or NR7, Y is O, and Z is CRnR12. OFG1 is preferably
Figure imgf000165_0001
attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (-CH2OFG1 in F or G). OFG2 is preferably attached directly to one of the carbons in the six-membered rings (-OFG2 in F or G). For both F and G, -CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-3; or vice versa. In certain embodiments, CH2OFG1 and OFG2 may be geminally substituted to one of the above-referenced carbons. The piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH2OFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. R" ' can be, e.g., C\-Ce alkyl, preferably CH3. The tethering attachment point is preferably nitrogen in both F and G.
[0323] In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH2; Y is CR9R10; Z is CRnR12, and R5 and R11 together form C6 cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH2; Y is CR9R10; Z is CRUR12, and R5 and R11 together form C5 cycloalkyl (H, z = 1). OFG1 is preferably attached to a primary carbon,
Figure imgf000166_0001
H e.g., an exocyclic methylene group (n=l) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [-(CF^OFG1 in H]. OFG2 is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG2 in H). -(CH^nOFG1 and OFG2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5.
Alternatively, -(CH^nOFG1 and OFG2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH2)nOFG1 may be attached to C-2 and OFG2 may be attached to C-3 ; -(CH^nOFG1 may be attached to C-3 and OFG2 may be attached to C-2; -(CH^nOFG1 may be attached to C-3 and OFG2 may be attached to C-4; or - (CH^nOFG1 may be attached to C-4 and OFG2 may be attached to C-3; -(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C-5; or -(CH^nOFG1 may be attached to C-5 and OFG2 may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. In a preferred embodiment, the substituents at C-l and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-l .
[0324] Other carriers may include those based on 3-hydroxyproline (J). Thus, -(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers
Figure imgf000167_0001
J and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.
[0325] Representative carriers are shown in FIG. 5.
[0326] In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether. Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any Ci-Cioo carbon-containing moiety, (e.g. C1-C75, C1-C50, C1-C20, C1-C10, C1-C6), preferably having at least one nitrogen atom. In preferred embodiments, the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand. Preferred tethers (underlined) include TAPZ
(CHANH?; TAP-qOYCH NH?; or
Figure imgf000167_0002
in which n is 1-6 and R" " is d- e alkyl. and Rd is hydrogen or a ligand. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or hydrazino group, -NHNH2. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP-(CH?)nNH(LIGAND) .
TAP-C(Q)(CH9)nNH(LIGAND). or TAP-NR' ' ' ' (CH?)nNH(LIGAND);
TAP-(CH?)nONH(LIGAND). TAP-C(0)(CH9)nONH(LIGAND). or
Figure imgf000167_0003
TAP-(CH?)nNHNH?(LIGAND).
TAP-C(0)(CH9)nNHNH9(LIGAND). or TAP-NR' ' "(CH9)nNHNH9(LIGAND).
[0327] In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position of the tether. Preferred electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred tethers (underlined) include TAPZ
(CH9)nCHO; TAP-C(Q)(CH9)nCHO; or TAP-NR" "(CH9)nCHO, in which n is 1-6 and R" " is Ci-C6 alkyl; or TAP-(CH9)nC(0)ONHS; TAP-C(Q)(CH9) nC(0)ONHS; or TAP-NR' ' "(C¾) nC(0)ONHS. in which n is 1-6 and R"" is Ci-C6 alkyl;
TAP-fCH CfOOC^Fs: TAP-CtOYCH?) Χ(Ό) OCF or TAP-NR" "(ΟΗ^ ηΟΓΟ OCfiF.. in which n is 1-6 and R"" is Ci-C6 alkyl; or -fCHACH7LG: TAP-CfOYCH CIfrLG: or TAP- NR""(CHACH7LG. in which n is 1-6 and R"" is Ci-C6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.
Tethered Entities
[0328] A wide variety of entities can be tethered to an iRNA agent, e.g., to the carrier of an RRMS. Examples are described below in the context of an RRMS but that is only preferred, entities can be coupled at other points to an iRNA agent.
[0329] Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a "precursor" RRMS after a "precursor" RRMS monomer has been incorporated into the growing strand. For example, an RRMS monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH2)nNH2 may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor RRMS by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor RRMS tether.
[0330] In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
[0331] Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides. [0332] Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.
[0333] Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a poly lysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L- lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
[0334] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic.
[0335] Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross- linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g.
EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
[0336] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactos amine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.
[0337] The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
[0338] The ligand can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin- 1 beta, or gamma interferon.
[0339] In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. Preferably, the target tissue is the liver, preferably parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a seru protein, e.g., HSA.
[0340] A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
[0341] In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non- kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
[0342] In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
[0343] In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).
[0344] In another aspect, the ligand is a cell-permeation agent, preferably a helical cell- permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
[0345] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three- dimensional structure similar to a natural peptide. The attachment of peptide and
peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
[0346] A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF. An RFGF analogue containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein and the Drosophila Antennapedia protein have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et ah, Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 50 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
[0347] An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et ah, Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et ah, Cancer Gene Therapy 8:783-787, 2001). The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing ayfi3 (Haubner et ah, Jour. Nucl. Med., 42:326-336, 2001).
[0348] Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an Iv¾ integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the Iv-¾ integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.
[0349] A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell- permeating peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., a -defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV- 1 gp41 and the NLS of SV40 large T antigen (Simeoni et ah, Nucl. Acids Res. 31 :2717-2724, 2003).
[0350] In one embodiment, a targeting peptide tethered to an RRMS can be an amphipathic a- helical peptide. Exemplary amphipathic a-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides, esculentinis- 1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomelic units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H- bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i ± 3, or i ± 4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.
[0351] Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
Methods for making iRNA agents
[0352] iRNA agents can include modified or non-naturally occuring bases, e.g., bases described in copending and coowned United States Provisional Application Serial No.
60/463,772 (Attorney Docket No. 14174-070P01), filed on April 17, 2003, which is hereby incorporated by reference and/or in copending and coowned United States Provisional
Application Serial No. 60/465,802 (Attorney Docket No. 14174-074P01), filed on April 25, 2003, which is hereby incorporated by reference. Monomers and iRNA agents which include such bases can be made by the methods found in United States Provisional Application Serial No. 60/463,772 (Attorney Docket No. 14174-070P01), filed on April 17, 2003, and/or in United States Provisional Application Serial No. 60/465,802 (Attorney Docket No. 14174-074P01), filed on April 25, 2003.
[0353] In addition, the invention includes iRNA agents having a modified or non-naturally occuring base and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a modified or non-naturally occuring base.
[0354] The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan, "Oligonucleotide Conjugates in Antisense Technology," in Antisense Drug
Technology, ed. S.T. Crooke, Marcel Dekker, Inc., 2001.
[0355] In one embodiment of the invention, a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide. For example, the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the a-carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain. For example, the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a "carbonyl donor," e.g., phenylchloroformate.
[0356] In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an N-methyl peptide. N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage. N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21 :99, 2000; Cell Penetrating Peptides: Processes and
Applications, Langel, ed., CRC Press, Boca Raton, FL, 2002; Fische et ah, Bioconjugate. Chem. 12: 825, 2001; Wander et ah, J. Am. Chem. Soc, 124: 13382, 2002). For example, an Ant or Tat peptide can be an N-methyl peptide.
[0357] In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a β-peptide. β-peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state, β-peptides are resistant to degradation by proteolytic enzymes, β-peptides can be synthesized by methods known in the art. For example, an Ant or Tat peptide can be a β-peptide.
[0358] In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate. Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters. For example, an Ant or Tat peptide can be an oligocarbamate.
[0359] In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an oligourea conjugate (or an oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety.
Replacement of the amide bond provides increased resistance to degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea conjugate is tethered to an iRNA agent for use in oral delivery. The backbone in each repeating unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid. The single carbon atom extension can increase peptide stability and lipophilicity, for example. An oligourea peptide can therefore be advantageous when an iRNA agent is directed for passage through a bacterial cell wall, or when an iRNA agent must traverse the blood-brain barrier, such as for the treatment of a neurological disorder. In one embodiment, a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor. For example, an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate).
[0360] The siRNA peptide conjugates of the invention can be affiliated with, e.g., tethered to, RRMSs occurring at various positions on an iRNA agent. For example, a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand). In another option, the peptide can be internally conjugated, such as in the loop of a short hairpin iRNA agent. In yet another option, the peptide can be affiliated with a complex, such as a peptide-carrier complex.
[0361] A peptide-carrier complex consists of at least a carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type. A carrier complex can carry additional targeting molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery. The one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier.
[0362] A carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer. A peptide moiety can be tethered to the carrier molecule by a variety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage. For example, a peptide-based linkage can be a GFLG peptide. Certain linkages will have particular advantages, and the advantages (or disadvantages) can be considered depending on the tissue target or intended use. For example, peptide based linkages are stable in the blood stream but are susceptible to enzymatic cleavage in the lysosomes.
Targeting
[0363] The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incorporation of an RRMS containing a ligand that targets the liver. For example, a liver-targeting agent can be a lipophilic moiety. Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine.
[0364] An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.
[0365] A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or mannose-6- phosphate targeting moiety can be used for macrophage targeting.
[0366] Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to the liver.
[0367] An iRNA agent targeted to the liver by an RRMS targeting moiety described herein can target a gene expressed in the liver. For example, the iRNA agent can target
p21(WAFl/DIPl), P27(KIP 1), the a-fetoprotein gene, beta-catenin, or c-MET, such as for treating a cancer of the liver. In another embodiment, the iRNA agent can target apoB-100, such as for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia; hypercholesterolemia; statin- resistant hypercholesterolemia; coronary artery disease (CAD); coronary heart disease (CHD); or atherosclerosis. In another embodiment, the iRNA agent can target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR- Gamma Coactivator (PGC-1); Fructose- 1 ,6-bisphosphatase; glucose-6-phosphatase; glucoses- phosphate translocator; glucokinase inhibitory regulatory protein; or phosphoenolpyruvate carboxykinase (PEPCK), such as to inhibit hepatic glucose production in a mammal, such as a human, such as for the treatment of diabetes. In another embodiment, an iRNA agent targeted to the liver can target Factor V, e.g., the Leiden Factor V allele, such as to reduce the tendency to form a blood clot. An iRNA agent targeted to the liver can include a sequence which targets hepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). For example, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.
[0368] Preferred ligands on RRMSs include folic acid, glucose, cholesterol, cholic acid, Vitamin E, Vitamin K, or Vitamin A.
Definitions
[0369] The term "halo" refers to any radical of fluorine, chlorine, bromine or iodine.
[0370] The term "alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term
"haloalkyl" refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S. The terms "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "aralkyl" include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.
[0371] The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. The sp2 and sp3 carbons may optionally serve as the point of attachment of the alkenyl and alkynyl groups, respectively.
[0372] The term "alkoxy" refers to an -O-alkyl radical. The term "aminoalkyl" refers to an alkyl substituted with an aminoThe term "mercapto" refers to an -SH radical. The term
"thioalkoxy" refers to an -S-alkyl radical.
[0373] The term "alkylene" refers to a divalent alkyl (i.e., -R-), e.g., -CH2-, -CH2CH2-, and - CH2CH2CH2-. The term "alkylenedioxo" refers to a divalent species of the structure -O-R-O-, in which R represents an alkylene.
[0374] The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted by a substituent. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl.
[0375] The term "cycloalkyl" as employed herein includes saturated cyclic, bicyclic, tricycliCjOr polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom capable of substitution can be substituted by a substituent. The cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon- carbon bond. Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and norbornyl.
[0376] The term "heterocyclyl" refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent. The heterocyclyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.
[0377] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent.
[0378] The term "oxo" refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.
[0379] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.
[0380] The term "substituents" refers to a group "substituted" on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO3H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(0)nalkyl (where n is 0-2), S(0)n aryl (where n is 0-2), S(0)n heteroaryl (where n is 0-2), S(0)n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted
heterocyclyl, and unsubstituted cycloalkyl. In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned substituents. [0381] The terms "adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl" and the like refer to radicals of adenine, cytosine, guanine, thymine, and uracil.
[0382] As used herein, an "unusual" nucleobase can include any one of the following:
2-methyladeninyl,
N6-methyladeninyl,
2-methylthio-N6-methyladeninyl,
N6-isopentenyladeninyl,
2-methylthio-N6-isopentenyladeninyl,
N6-(cis-hydroxyisopentenyl)adeninyl,
2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl,
N6-glycinylcarbamoyladeninyl,
N6-threonylcarbamoyladeninyl,
2-methylthio-N6-threonyl carbamoyladeninyl,
N6-methyl-N6-threonylcarbamoyladeninyl,
N6-hydroxynorvalylcarbamoyladeninyl,
2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl,
N6,N6-dimethyladeninyl,
3 -methylcytosinyl,
5 -methylcytosinyl,
2-thiocytosinyl,
5-formylcytosinyl,
Figure imgf000179_0001
N4-methylcytosinyl,
5 -hydroxymethylcytosinyl,
1 -methylguaninyl,
N2-methylguaninyl,
7-methylguaninyl,
N2 ,N2 -dimethy lguaniny 1,
Figure imgf000180_0001
- 179- N2,N2,7-trimethylguaninyl,
1 -methylguaninyl,
7-cyano-7-deazaguaninyl,
7-aminomethyl-7-deazaguaninyl,
pseudouracilyl,
dihydrouracilyl,
5 -methy luracily 1,
1 -methy lpseudouracilyl,
2-thiouracilyl,
4- thiouracilyl,
2- thiothyminyl
5 -methy 1-2 -thiouracily 1,
3- (3-amino-3-carboxypropyl)uracilyl,
5 -hy droxyuracily 1,
5 -methoxyuracily 1,
uracilyl 5-oxyacetic acid,
uracilyl 5-oxyacetic acid methyl ester,
5- (carboxyhydroxymethyl)uracilyl,
5-(carboxyhydroxymethyl)uracilyl methyl ester, 5-methoxycarbonylmethyluracilyl,
5-methoxycarbonylmethyl-2-thiouracilyl,
5 -aminomethyl-2-thiouracilyl,
5 -methy laminomethy luracily 1,
5-methylaminomethyl-2-thiouracilyl,
5-methylaminomethyl-2-selenouracilyl,
5-carbamoylmethyluracilyl,
5-carboxymethylaminomethyluracilyl,
5-carboxymethylaminomethyl-2-thiouracilyl,
3 -methy luracily 1,
l-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl, 5 -carboxymethyluracilyl,
5-methyldihydrouracilyl, or
3 -methylpseudouracilyl. Asymmetrical Modifications
[0383] In one aspect, the invention features an iRNA agent which can be asymmetrically modified as described herein.
[0384] In addition, the invention includes iRNA agents having asymmetrical modifications and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates an asymmetrical modification.
[0385] iRNA agents of the invention can be asymmetrically modified. An asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand. An asymmetrical modification is a modification found on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification. An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein. E.g., an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue; modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway. The designation of a modification as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting.
[0386] While not wishing to be bound by theory or any particular mechanistic model, it is believed that asymmetrical modification allows an iRNA agent to be optimized in view of the different or "asymmetrical" functions of the sense and antisense strands. For example, both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense strand . In addition, since some modifications, e.g., targeting moieties, can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such modifications are preferably placed on the sense strand. Thus, targeting moieties, especially bulky ones (e.g. cholesterol), are preferentially added to the sense strand. In one embodiment, an asymmetrical modification in which a phosphate of the backbone is substituted with S, e.g., a phosphorothioate modification, is present in the antisense strand, and a 2' modification, e.g., 2' OMe is present in the sense strand. A targeting moiety can be present at either (or both) the 5' or 3' end of the sense strand of the iRNA agent. In a preferred example, a P of the backbone is replaced with S in the antisense strand, 2'OMe is present in the sense strand, and a targeting moiety is added to either the 5' or 3' end of the sense strand of the iRNA agent.
[0387] In a preferred embodiment an asymmetrically modified iRNA agent has a
modification on the sense strand which modification is not found on the antisense strand and the antisense strand has a modification which is not found on the sense strand.
[0388] Each strand can include one or more asymmetrical modifications. By way of example: one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA. E.g., one strand, e.g., the sense strand can have a modification which targets the iRNA agent to a tissue, and the other strand, e.g., the antisense strand, has a modification which promotes hybridization with the target gene sequence.
[0389] In some embodiments both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well. E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand.
[0390] In an embodiment one strand has an asymmetrical 2' modification, e.g., a 2' OMe modification, and the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification. So, in one embodiment the antisense strand has an asymmetrical 2' OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa). In a particularly preferred embodiment the RNAi agent will have asymmetrical 2'-0 alkyl, preferably, 2 '-OMe modifications on the sense strand and
asymmetrical backbone P modification, preferably a phosphothioate modification in the antisense strand. There can be one or multiple 2'-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the antisense strand can be so modified. It is preferable to have an iRNA agent wherein there are multiple 2'- OMe modifications on the sense strand and multiple phophorothioate modifications on the antisense strand. All of the subunits on one or both strands can be so modified. A particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region about 20-21, and preferably 19, subunits in length and one or two 3 ' overhangs of about 2 subunits in length. [0391] Asymmetrical modifications are useful for promoting resistance to degradation by nucleases, e.g., endonucleases. iRNA agents can include one or more asymmetrical
modifications which promote resistance to degradation. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Most if not all sites on a strand are vulnerable, to some degree, to degradation by endonucleases. One can determine sites which are relatively vulnerable and insert asymmetrical modifications which inhibit degradation. It is often desirable to provide asymmetrical modification of a UA site in an iRNA agent, and in some cases it is desirable to provide the UA sequence on both strands with asymmetrical modification. Examples of modifications which inhibit endonucleolytic degradation can be found herein. Particularly favored modifications include: 2' modification, e.g., provision of a 2' OMe moiety on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement of the U with a C5 amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2', 6', 7', or 8' position. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
[0392] Asymmetrical modification can be used to inhibit degradation by exonucleases.
Asymmetrical modifications can include those in which only one strand is modified as well as those in which both are modified. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3 ' overhang, e.g., at the 3 ' terminus, and on the antisense strand, e.g., in a 3' overhang, e.g., at the 3' terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the
modifications introduce moieties of different charge it is preferable that the one with greater charge be on the sense strand.
[0393] Examples of modifications which inhibit exonucleolytic degradation can be found herein. Particularly favored modifications include: 2' modification, e.g., provision of a 2' OMe moiety in a 3 ' overhang, e.g., at the 3' terminus (3 ' terminus means at the 3 ' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P in a 3 ' overhang, e.g., at the 3 ' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and
modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3 ' overhang, e.g., at the 3 ' terminus; modification with a 3' alkyl; modification with an abasic pyrolidine in a 3 ' overhang, e.g., at the 3 ' terminus; modification with naproxene, ibuprofen, or other moieties which inhibit degradation at the 3' terminus. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
[0394] Modifications, e.g., those described herein, which affect targeting can be provided as asymmetrical modifications. Targeting modifications which can inhibit silencing, e.g., by inhibiting cleavage of a target, can be provided as asymmetrical modifications of the sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand. Targeting modifications which introduce moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive charge or one negative charge) can be placed on the sense strand.
[0395] Modifications, e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical
modifications. These include: 5 methyl U; 5 methyl C; pseudouridine, Locked nucleic acids ,2 thio U and 2-amino-A. In some embodiments one or more of these is provided on the antisense strand.
[0396] iRNA agents have a defined structure, with a sense strand and an antisense strand, and in many cases short single strand overhangs, e.g., of 2 or 3 nucleotides are present at one or both 3 ' ends. Asymmetrical modification can be used to optimize the activity of such a structure, e.g., by being placed selectively within the iRNA. E.g., the end region of the iRNA agent defined by the 5' end of the sense strand and the 3 'end of the antisense strand is important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3 ' overhang. In preferred embodiments asymmetrical modifications which result in one or more of the following are used: modifications of the 5' end of the sense strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5 ' exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a "tight" structure at this end of the molecule.
[0397] The end region of the iRNA agent defined by the 3' end of the sense strand and the 5 'end of the antisense strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3 ' overhang. Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an "open" structure at this end of the molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification of the antisense strand which inhibit kinase activation are avoided in preferred embodiments.
[0398] Exemplary modifications for asymmetrical placement in the sense strand include the following:
(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S-P=S); these modifications can be used to promote nuclease resistance;
(b) 2'-0 alkyl, e.g., 2'-OMe, 3'-0 alkyl, e.g., 3'-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand, the 3' modifications can be used at the 5' end of the sense strand to avoid sense strand activation by RISC;
(c) 2 '-5' linkages (with 2'-H, 2' -OH and 2'-OMe and with P=0 or P=S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC;
(d) L sugars (e.g., L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC;
(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5 ' end of the sense strand to avoid sense strand activation by RISC;
(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand;
(g) cationic groups and Zwitterionic groups (preferably at a terminus), these modifications can be used to promote nuclease resistance;
(h) conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC.
[0399] Exemplary modifications for asymmetrical placement in the antisense strand include the following:
(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S-P=S);
(b) 2'-0 alkyl, e.g., 2'-OMe, (at terminal positions);
(c) 2'-5' linkages (with 2'-H, 2' -OH and 2'-OMe) e.g., terminal at the 3' end); e.g., with P=0 or P=S preferably at the 3 '-end, these modifications are preferably excluded from the 5 ' end region as they may interfere with RISC enzyme activity such as kinase activity;
(d) L sugars (e.g, L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe); e.g., terminal at the 3' end; e.g., with P=0 or P=S preferably at the 3 '-end, these modifications are preferably excluded from the 5 ' end region as they may interfere with kinase activity;
(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modifications are preferably excluded from the 5' end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a "loose" structure in the 5' region, additionally, they may interfere with kinase activity;
(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines);
(g) cationic groups and Zwitterionic groups (preferably at a terminus);
conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred.
[0400] The 5'-OH of the antisense strand should be kept free to promote activity. In some preferred embodiments modifications that promote nuclease resistance should be included at the 3 ' end, particularly in the 3 ' overhang.
[0401] In another aspect, the invention features a method of optimizing, e.g., stabilizing, an iRNA agent. The method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity.
[0402] The decrease in activity can be less than a preselected level of decrease. In preferred embodiments decrease in activity means a decrease of less than 5, 10, 20, 40, or 50 % activity, as compared with an otherwise similar iRNA lacking the introduced modification. Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the required maintenance of activity. [0403] The optimized property can be any property described herein and in particular the properties discussed in the section on asymmetrical modifications provided herein. The modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein. Particularly preferred asymmetric modifications are 2'-0 alkyl modifications, e.g., 2'-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate modifications, in the antisense sequence.
[0404] In a preferred embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications.
[0405] Multiple asymmetric modifications can be introduced into either or both of the sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified.
Z-X-Y Architecture
[0406] In one aspect, the invention features an iRNA agent which can have a Z-X-Y architecture or structure such as those described herein and those described in copending, co- owned United States Provisional Application Serial No. 60/510,246 (Attorney Docket No. 14174-079P02), filed on October 9, 2003, which is hereby incorporated by reference, and in copending, co-owned United States Provisional Application Serial No. 60/510,318 (Attorney Docket No. 14174-079P03), filed on October 10, 2003, which is hereby incorporated by reference.
[0407] In addition, the invention includes iRNA agents having a Z-X-Y structure and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a Z-X-Y architecture.
[0408] The invention provides an iRNA agent having a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region:
Z— X— Y. [0409] It may be desirable to modify subunits in one or both of Zand/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X.
[0410] The Z region typically includes a terminus of an iRNA agent. The length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some embodiments self associate, e.g., to form a loop structure. Such structures can be formed by the end of a strand looping back and forming an intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand. A typical embodiment of a Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein. The Z region can thus be or include a 3 ' or 5 ' terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3- overhang. Typical inter-subunit bonds in the Z region include: P=0; P=S; S-P=S; P-NR2; and P-BR2. Chiral P=X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.) Other preferred Z region subunit modifications (also discussed elsewhere herein) can include: 3 '-OR, 3'SR, 2'-OMe, 3'-OMe, and 2ΌΗ
modifications and moieties; alpha configuration bases; and 2' arabino modifications.
[0411] The X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand. The length of the X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits. Particularly preferred region X's will include 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengths are described elsewhere herein and can be used. Typical X region subunits include 2'-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phophorothioate or non-phosphate bonds are absent. Other modifications preferred in the X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines. Some embodiments have 4 or more consecutive 2ΌΗ subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2ΌΗ subunits.
[0412] The Y region will generally conform to the the parameters set out for the Z regions. However, the X and Z regions need not be the same, different types and numbers of
modifications can be present, and infact, one will usually be a 3' overhang and one will usually be a 5' overhang. [0413] In a preferred embodiment the iRNA agent will have a Y and/or Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe or other alkyl; and an X region that includes at least four consecutive ribonucleoside subunits in which the 2'-OH remains unsubstituted.
[0414] The subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage. These
modifications can be provided bewteen the subunits of any of the regions, Y, X, and Z.
However, it is preferred that their occureceis minimized and in particular it is preferred that consecutive modified linkages be avoided.
[0415] In a preferred embodiment the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe; and an X region that includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2' -OH remains unsubstituted.
[0416] As mentioned above, the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in particular it is preferred that consecutive modified linkages be avoided.
[0417] Thus, in a preferred embodiment, not all of the subunit linkages of the iRNA agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred iRNA agents will not have four or more consecutive subunits, e.g., 2'-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.
[0418] It is particularly preferred to minimize the occurrence in region X. Thus, in preferred embodiments each of the nucleoside subunit linkages in X will be phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized. E.g., although the Y and/or Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized. Thus, in preferred embodiments the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred X regions will not have four or more consecutive subunits, e.g., 2'-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA. [0419] In a preferred embodiment Y and /or Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications of the subunit linkages.
[0420] In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2' moieties.
[0421] In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages.
[0422] In a preferred embodiment one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., a 2'-0-alkyl moiety. E.g., any adjacent 2'-0-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosphorothioate linkage.
[0423] In a preferred embodiment each of Y and/or Z independently has only one
phosphorothioate linkage between adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides. If there is a second set of adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides, in Y and/or Z that second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.
[0424] In a prefered embodiment each of Y and/orZ independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides, are be connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.
[0425] In a prefered embodiment one of the above recited limitation on adjacent subunits in Y and or Z is combined with a limitation on the subunits in X. E.g., one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., a 2'-0-alkyl moiety. E.g., any adjacent 2'-0-alkyl moieties in the Y and/or Z, are connected by a linkage other than a phosporothioate linkage. In addition, the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2' moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages.
[0426] A Y and/or Z region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can be present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively. In a preferred embodiment the frequency of the modification will differ between Y and /or Z and X, e.g., the modification will be present one of Y and/or Z or X and absent in the other.
[0427] An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification desribed herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can b present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.
[0428] An RRMS (described elswhere herein) can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed in a Y and/or Z region, at or near (within 1, 2, or 3 positions) of the 3 ' or 5' end of the sense strand or at near (within 2 or 3 positions of) the 3 ' end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end of the antisense strand. An RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical for antisense binding to the target.
Differential Modification of Terminal Duplex Stability
[0429] In one aspect, the invention features an iRNA agent which can have differential modification of terminal duplex stability (DMTDS).
[0430] In addition, the invention includes iRNA agents having DMTDS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS.
[0431] iRNA agents can be optimized by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5' end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5' end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5 'end . While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5' end of the antisense strand.
[0432] The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex association), in the region of the 3 ' end of the antisense strand duplex. This can be
accomplished, e.g., by the inclusion of subunits which decrease the propensity of the duplex to disassociate or melt in the region of the 3' end of the antisense strand. It can also be
accomplished by the attachment of ligand that decreases the propensity of the duplex to disassociate of melt in the region of the 5'end.
[0433] Modifications which increase the tendency of the 5' end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3' end of the duplex to dissociate. Likewise, modifications which decrease the tendency of the 3' end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5' end of the duplex to dissociate.
Decreasing the stability of the AS 5 ' end of the duplex
[0434] Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation:
A:U is preferred over G:C;
G:U is preferred over G:C;
I:C is preferred over G:C (I=inosine);
mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings;
pairings which include a universal base are preferred over canonical pairings. [0435] A typical ds iR A agent can be diagrammed as follows: s 5' Ri Ni N2 N3 N4 N5
Figure imgf000194_0001
AS 3 ' R3 i N2 N3 N4 N5
Figure imgf000194_0002
S:AS Pi P2 P3 P4
P5 [N] P-5 P-4 P-3 P-2 P-i
5'
[0436] S indicates the sense strand; AS indicates antisense strand; Ri indicates an optional (and nonpreferred) 5' sense strand overhang; R2 indicates an optional (though preferred) 3' sense overhang; R3 indicates an optional (though preferred) 3 ' antisense sense overhang; R4 indicates an optional (and nonpreferred) 5' antisense overhang; N indicates subunits; [N] indicates that additional subunit pairs may be present; and Px, indicates a paring of sense Nx and antisense Nx. Overhangs are not shown in the P diagram. In some embodiments a 3 ' AS overhang corresponds to region Z, the duplex region corresponds to region X, and the 3 ' S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.)
[0437] It is preferred that pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5' end of the AS strand. The terminal pair (the most 5' pair in terms of the AS strand) is designated as P_i, and the subsequent pairing positions (going in the 3 ' direction in terms of the AS strand) in the duplex are designated, P-2, P-3, P_4, P-5, and so on. The preferred region in which to modify to modulate duplex formation is at P_5 through P_i, more preferably P_4 through P_i , more preferably P-3 through P_i. Modification at P_ 1, is particularly preferred, alone or with modification(s) other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of:
A:U
G:U
I:C
mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base. [0438] In preferred embodiments the change in subunit needed to achieve a pairing which promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand.
[0439] In a preferred embodiment the at least 2, or 3, of the pairs in P_i, through P_4, are pairs which promote disociation.
[0440] In a preferred embodiment the at least 2, or 3, of the pairs in P_i, through P_4, are A:U.
[0441] In a preferred embodiment the at least 2, or 3, of the pairs in P_i, through P_4, are G:U.
[0442] In a preferred embodiment the at least 2, or 3, of the pairs in P_i, through P_4, are I:C.
[0443] In a preferred embodiment the at least 2, or 3, of the pairs in P_i, through P_4, are mismatched pairs, e.g., non-canonical or other than canonical pairings pairings.
[0444] In a preferred embodiment the at least 2, or 3, of the pairs in P_i, through P_4, are pairings which include a universal base.
Increasing the stability of the AS 3 ' end of the duplex
[0445] Subunit pairs can be ranked on the basis of their propensity to promote stability and inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting duplex stability:
G:C is preferred over A:U
Watson-Crick matches (A:T, A:U, G:C) are preferred over non-canonical or other than canonical pairings
analogs that increase stability are preferred over Watson-Crick matches (A:T, A:U, G:C) 2-amino-A:U is preferred over A:U
2-thio U or 5 Me-thio-U:A are preferred over U:A
G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G guanadinium-G-clamp:G is preferred over C:G
psuedo uridine:A is preferred over U:A
sugar modifications, e.g., 2' modifications, e.g., 2'F, ENA, or LNA, which enhance binding are preferred over non-modified moieties and can be present on one or both strands to enhance stability of the duplex. It is preferred that pairings which increase the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 3 ' end of the AS strand. The terminal pair (the most 3 ' pair in terms of the AS strand) is designated as Pi, and the subsequent pairing positions (going in the 5' direction in terms of the AS strand) in the duplex are designated, P2, P3, P4, P5, and so on. The preferred region in which to modify to modulate duplex formation is at P5 through Pi, more preferably P4 through Pi , more preferably P3 through Pi. Modification at Pi, is particularly preferred, alone or with mdification(s) at other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of the recited regions be chosen independently from the group of:
G:C
a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U,
G:C)
2-amino-A:U
2-thio U or 5 Me-thio-U:A
G-clamp (an analog of C having 4 hydrogen bonds):G
guanadinium-G-clamp : G
psuedo uridine:A
a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhance binding.
[0446] In a preferred embodiment the at least 2, or 3, of the pairs in P_i, through P_4, are pairs which promote duplex stability.
[0447] In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are G:C.
[0448] In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are a pair having an analog that increases stability over Watson-Crick matches.
[0449] In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are 2- amino-A:U.
[0450] In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are 2-thio U or 5 Me-thio-U:A.
[0451] In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are G- clamp:G.
[0452] In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are guanidinium-G-clamp:G.
[0453] In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are psuedo uridine:A.
[0454] In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhances binding.
[0455] G-clamps and guanidinium G-clamps are discussed in the following references:
Holmes and Gait, "The Synthesis of 2'-0-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition of the HIV-1 Tat-TAR Interaction," Nucleosides, Nucleotides & Nucleic Acids, 22: 1259-1262, 2003; Holmes et al., "Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2'-0-methyl G-clamp ribonucleoside analogues," Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al., "Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue:
Guanidinium G-clamp," Helvetica Chimica Acta, 86:966-978, 2003; Rajeev, et al., "High- Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues," Organic Letters, 4:4395-4398, 2002; Ausin, et al, "Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers," Organic Letters, 4:4073-4075, 2002; Maier et al., "Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2- aminoethoxy)-phenoxazine ("G-clamp") and origins of their nuclease resistance properties," Biochemistry, 41 : 1323-7, 2002; Flanagan, et al., "A cytosine analog that confers enhanced potency to antisense oligonucleotides," Proceedings Of The National Academy Of Sciences Of The United States Of America, 96:3513-8, 1999.Gclamps may also comprise a locked nucleic acid. Methods of synthesizing such LNA-Gclamps are shown in the following schematics.
Figure imgf000198_0001
[0456] Compound 900 is synthesized utilizing the literature procedure (Tetrahedron Lett., 49, 7168, 2008). Treatment of 900 with acetic anhydride in pyridine followed by reaction with N- bromosuccinimide gives the protected 5-bromo LNA 902. This on reaction with phosphorus oxychloride and 1,2,4-triazole in acetonitrile gives the triazolyl derivative 903 which on treatment with aminophenol is converted to the corresponding N-substituted cytidine derivative 904 . Cyclization of 904 is accomplished by treating with triethylamine in ethanol to give the protected LNA- Phenoxazine or G-Clamp. Removal of the protecting groups is accomplished by treating with ammonium hydroxide at room temperature to give 906. This on reaction with DMTr-Cl gives the 5'-DMTr derivative 907 which on phosphitylation gives the target phosphoramidite 908.
[0457] The dimethoxytrityl derivative 907 is converted to the corresponding succinate 909 by the treatment with succinic anhydride in pyridine in the presence of DMAP. This is activated using HBTU in DMF in the presence of Hunig's base and the activated ester is coupled with long chain alkyl amine-CPG to give 910.
Pyrrolopyrimidine Gclamp.
Figure imgf000200_0001
[0459] Compound 225A: To a suspension of 223A (lOg) in dry methanol (20 ml) was added ICl (lOg) and the mixture was heated under reflux for 20 h. The solvent was evaporated and the crude was purified by silica gel column chromatography using a gradient 0-25% methanol in dichloromethane to give 1 1.2 g of 224A. To a cold solution of 224A (1 g, 2.69 mmol) in dry pyridine (20 ml) was added benzoyl chloride (1.26 ml, 10.8 mmol) and the mixture was stirred at room temperature overnight. Reaction was quenched by the addition of water. The mixture was diluted with dichloromethane and washed with sat. sodium bicarbonate solution. Organic layer was evaporated and coevaporated with toluene. The residue was purified by silica gel column chromatography using a gradient 0-5% methanol in dichloromethane to give 1.6 g of 225A.
[0460] Compound 221 K: A solution of 225A (4.2g, 6.1 4mmol) and 226 (2.3 g, 8.8 mmol) in anhydrous DMF (60 ml) and triethylamine (30 ml) was degassed by bubbling argon. To this solution Dichloro bis(triphenylphosphine)palladium (0.42 g) and copper iodide (0.23 g) were added and the mixture was heated at 48°C for 18h. The solvent was evaporated and the residue was dissolved in methanol (300 ml). The reaction mixture was heated under reflux for 18 h. The solvent was evaporated and the residue was purified by silica gel column chromatography. The product was eluted using a gradient of 0-5% methanol in dichloromethane. Evaporation of the appropriate fractions containing the product gave 3.8 g of 227 A.
[0461] Compound 228A: To a cold (ice bath) solution of 227A (3.8 g) in a mixture of pyridine (60 ml) and methanol (60 ml) was added 1 N NaOH (1 1 ml). After stirring the mixture at 0-5°C for lh, the reaction was quenched by the addition of dilute HCl (IN, 11 ml). Methanol was evaporated and the solution was diluted with dichloromethane (250 ml) and washed with water (50 ml). Organic layer was dried over sodium sulfate and evaporated. The residue was co- evaporated with toluene and purified by silica gel column chromatography to give 1.8 g of 228A.
[0462] ¾ NMR (400 MHz, DMSO) δ 1 1.47 (s, 1H), 8.71 (s, 1H), 7.78 - 7.66 (m, 1H), 7.38 - 7.25 (m, 1H), 7.1 1 (t, J= 6.6, 2H), 7.00 (t, J= 7.5, 1H), 6.76 (s, 1H), 6.04 (d, J= 17.5, 1H), 5.57 (d, J= 6.5, 1H), 5.29 (t, J= 5.0, 1H), 4.93 (dd, J= 52.9, 4.0, 1H), 4.22 - 4.05 (m, 3H), 3.97 (d, J = 8.7, 1H), 3.94 - 3.82 (m, 1H), 3.75 - 3.62 (m, 1H), 3.38 (dd, J= 1 1.1, 5.5, 2H), 1.34 (s, 9H).
[0463] 19F NMR (376 MHz, DMSO) δ -203.50 (m).
[0464] MS: Calcd: 505 Found:504 (M-l)-
[0465] Compound 229A: A solution of compound 228A (0.8g) in 50% TFA-dichloromethane (20 ml) was stirred at 0°C for 2h. After this time the reaction mixture was evaporated. The residue was co-evaporated with toluene (25 ml) followed by anhydrous pyridine (20 ml). This was dissolved in dry pyridine (15 ml), the solution was cooled in an ice bath and trifluoroacetic anhydride (2 ml) was added. The reaction mixture was stirred at 0-5°C for 2h. Reaction was quenched by the addition of methanol, diluted with dichloromethane (150 ml) and washed with water (50 ml). Organic layer was dried over sodium sulfate and evaporated. The residue was co- evaporated with toluene (20 ml) and purified by silica gel column chromatography. The product was eluted using a gradient of 0-10% methanol in dichloromethane. Appropriate fractions containing the product were evaporated to give 0.4 g of pure 229A. [0466] Compound 230A: To a solution of 229A (0.4 g) in anhydrous pyridine was added 4,4'-DMT-Cl ( 0.4 g) and the mixture was stirred at room temperature for 5h. The reaction mixture was diluted with dichloromethane (100 ml) and washed with water (50 ml). Organic layer was evaporated and the residue was co-evaporated with toluene. The product was purified using silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane to give 0.36 g of 230A.
[0467] ¾ NMR (400 MHz, DMSO) δ 1 1.39 (s, 1H), 9.57 (t, J= 5.3, 1H), 8.54 (s, 1H), 7.64 - 7.54 (m, 1H), 7.43 (d, J= 7.5, 3H), 7.36 - 7.26 (m, 9H), 7.21 (t, J= 7.2, 1H), 7.13 (d, J= 8.3, 1H), 7.02 (t, J= 7.6, 1H), 6.86 (dd, J= 8.9, 7.4, 5H), 6.02 (d, J= 18.6, 1H), 5.77 - 5.68 (m, 1H), 5.01 (dd, J= 52.9, 3.9, 1H), 4.55 - 4.37 (m, 1H), 4.14 (dd, J= 13.3, 7.4, 4H), 4.00 (q, J= 7.1, 1H), 3.70 - 3.60 (m, 8H), 3.57 - 3.42 (m, 4H), 3.34 (d, J= 1 1.1, 2H).
[0468] 19F NMR (376 MHz, DMSO) δ -77.22 (s), -202.65 (m).
[0469] Compound 230 B (2'OMe PC-GClamp): In a similar manner as described for 230A, compound 230B was synthesized starting from Tri-benzoyl-2'-OMe-5-iodocytidine and 226.
[0470] ¾ NMR (400 MHz, DMSO) δ 1 1.35 (s, 1H), 9.56 (t, J= 5.4, 1H), 8.57 (s, 1H), 7.64 - 7.54 (m, 1H), 7.43 (d, J= 7.5, 2H), 7.38 - 7.26 (m, 8H), 7.21 (t, J= 7.2, 1H), 7.13 (d, J= 8.3, 1H), 7.02 (t, J= 7.5, 1H), 6.86 (t, J= 8.2, 4H), 5.94 (s, 1H), 5.87 (s, 1H), 5.22 (d, J= 7.5, 1H), 4.32 (td, J= 8.1, 5.1, 1H), 4.14 (t, J= 5.5, 2H), 4.06 (d, J= 7.5, 1H), 3.73 (d, J= 4.8, 1H), 3.66 (s, 3H), 3.63 (s, 3H), 3.53 (s, 3H).
[0471] 19F NMR (376 MHz, DMSO) δ -77.15 (s).
[0472] Compound 231 : To a solution of 230 (lmmol) in dichloromethane (10ml) is added 2- cyanoethyl-tetraisopropylphosphoramidite (1.3 mmol) and dicyanoimidazole (0.9 mmol). The mixture is stirred at room temperature for 6 h, diluted with dichloromethane and washed with sodium bicarbonate solution. Organic layer is dried over sodium sulfate and evaporated. The residue is subjected to column chromatography to give compound 231.
[0473] Solid support derivatization: The dimethoxytrityl derivative 230 is converted to the corresponding succinate 232 by the treatment with succinic anhydride in pyridine in the presence of DMAP. This is activated using HBTU in DMF in the presence of Hunig's base and the activated ester is coupled with long chain alkyl amine-CPG to give 232B.
Figure imgf000203_0001
[0474] Tribenzoyl-5-iodocytidine 235 on reaction with 3 -ethynylpyridine (or 4- ethynylpyridine) in DMF in the presence of bis(triphenylphosphine)palladium(II) dichloride and Cul gave 236. Deprotection of 236 with sodium hydroxide in a mixture of methanol and pyridine gave 237 which on reaction with DMTr-Cl in pyridine gives the corresponding 5'- dimethoxytrityl derivative 238. This on phosphitylation with 2-cyanoethyl N,N- diisopropylchlorophosphoramidite in dichloromethane in the presence of Hunig's base gives the target monomer 239.
[0475] Derivatization of the solid support: The dimethoxytrityl derivative 238 is converted to the corresponding succinate 240 by the treatment with succinic anhydride in pyridine in the presence of DMAP. This is activated using HBTU in DMF in the presence of Hunig's base and the activated ester is coupled with long chain alkyl amine-CPG to give 241.
[0476] Simultaneously decreasing the stability of the AS 5'end of the duplex and increasing the stability of the AS 3 ' end of the duplex
[0477] As is discussed above, an iRNA agent can be modified to both decrease the stability of the AS 5'end of the duplex and increase the stability of the AS 3' end of the duplex. This can be effected by combining one or more of the stability decreasing modifications in the AS 5' end of the duplex with one or more of the stability increasing modifications in the AS 3' end of the duplex. Accordingly a preferred embodiment includes modification in P_5 through P_i, more preferably P_4 through P_i and more preferably P_3 through P_i. Modification at P_i, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 5' end of the duplex region be chosen independently from the group of:
A:U
G:U
I:C
mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and a modification in P5 through Pi, more preferably P4 through Pi and more preferably P3 through Pi. Modification at Pi, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 3' end of the duplex region be chosen independently from the group of:
G:C
a pair having an analog that increases stability over Watson-Crick matches (A:T,
A:U, G:C)
2-amino-A:U 2-thio U or 5 Me-thio-U:A
G-clamp (an analog of C having 4 hydrogen bonds):G
guanadinium-G-clam : G
psuedo uridine:A
a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhance binding.
[0478] The invention also includes methods of selecting and making iRNA agents having DMTDS. E.g., when screening a target sequence for candidate sequences for use as iRNA agents one can select sequences having a DMTDS property described herein or one which can be modified, preferably with as few changes as possible, especially to the
AS strand, to provide a desired level of DMTDS.
[0479] The invention also includes, providing a candidate iRNA agent sequence, and modifying at least one P in P_5 through P_i and/or at least one P in P5 through Pi to provide a DMTDS iRNA agent.
[0480] DMTDS iRNA agents can be used in any method described herein, e.g., to silence any gene disclosed herein, to treat any disorder described herein, in any formulation described herein, and generally in and/or with the methods and compositions described elsewhere herein. DMTDS iRNA agents can incorporate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of nuclease resistant monomers or the inclusion of single strand overhangs (e.g., 3 ' AS overhangs and/or 3' S strand overhangs) which self associate to form intrastrand duplex structure.
[0481] Preferably these iRNA agents will have an architecture described herein.
In vivo Delivery
[0482] An iRNA agent can be linked, e.g., noncovalently linked to a polymer for the efficient delivery of the iRNA agent to a subject, e.g., a mammal, such as a human. The iRNA agent can, for example, be complexed with cyclodextrin. Cyclodextrins have been used as delivery vehicles of therapeutic compounds. Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity of the cyclodextrin. In other examples, cyclodextrins form non-covalent associations with other biologically active molecules such as oligonucleotides and derivatives thereof. The use of cyclodextrins creates a water-soluble drug delivery complex, that can be modified with targeting or other functional groups. Cyclodextrin cellular delivery system for oligonucleotides described in U.S. Pat. No. 5,691,316, which is hereby incorporated by reference, are suitable for use in methods of the invention. In this system, an oligonucleotide is noncovalently complexed with a cyclodextrin, or the oligonucleotide is covalently bound to adamantine which in turn is non-covalently associated with a cyclodextrin.
[0483] The delivery molecule can include a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer having at least one ligand bound to the cyclodextrin copolymer. Delivery systems , as described in U.S. Patent No. 6,509,323, herein incorporated by reference, are suitable for use in methods of the invention. An iRNA agent can be bound to the linear cyclodextrin copolymer and/or a linear oxidized cyclodextrin copolymer. Either or both of the cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer and/or bound to a ligand.
[0484] A composition for iRNA delivery can employ an "inclusion complex," a molecular compound having the characteristic structure of an adduct. In this structure, the "host molecule" spatially encloses at least part of another compound in the delivery vehicle. The enclosed compound (the "guest molecule") is situated in the cavity of the host molecule without affecting the framework structure of the host. A "host" is preferably cyclodextrin, but can be any of the molecules suggested in U.S. Patent Publ. 2003/0008818, herein incorporated by reference in its entirety.
[0485] Cyclodextrins can interact with a variety of ionic and molecular species, and the resulting inclusion compounds belong to the class of "host-guest" complexes. Within the host- guest relationship, the binding sites of the host and guest molecules should be complementary in the stereoelectronic sense. A composition of the invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite of the polymer and therapeutic agent, e.g., the iRNA agent. The iRNA agent can contain one or more complexing agents. At least one polymer of the particulate composite can interact with the complexing agent in a host-guest or a guest-host interaction to form an inclusion complex between the polymer and the complexing agent. The polymer and, more particularly, the complexing agent can be used to introduce functionality into the composition. For example, at least one polymer of the particulate composite has host functionality and forms an inclusion complex with a complexing agent having guest functionality. Alternatively, at least one polymer of the particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer of the particulate composite can also contain both host and guest functionalities and form inclusion complexes with guest complexing agents and host complexing agents. A polymer with functionality can, for example, facilitate cell targeting and/or cell contact (e.g., targeting or contact to a liver cell), intercellular trafficking, and/or cell entry and release. [0486] Upon forming the particulate composite, the iRNA agent may or may not retain its biological or therapeutic activity. Upon release from the therapeutic composition, specifically, from the polymer of the particulate composite, the activity of the iRNA agent is restored.
Accordingly, the particulate composite advantageously affords the iRNA agent protection against loss of activity due to, for example, degradation and offers enhanced bioavailability. Thus, a composition may be used to provide stability, particularly storage or solution stability, to an iRNA agent or any active chemical compound. The iRNA agent may be further modified with a ligand prior to or after particulate composite or therapeutic composition formation. The ligand can provide further functionality. For example, the ligand can be a targeting moiety.
Physiological Effects
[0487] The iRNA agents described herein can be designed such that determining therapeutic toxicity is made easier by the complementarity of the iRNA agent with both a human and a non- human animal sequence. By these methods, an iRNA agent can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal, e.g., a non-human mammal, such as a rodent, ruminant or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus, Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence of the iRNA agent could be complementary to sequences within homologous genes, e.g., oncogenes or tumor suppressor genes, of the non-human mammal and the human. By determining the toxicity of the iRNA agent in the non-human mammal, one can extrapolate the toxicity of the iRNA agent in a human. For a more strenuous toxicity test, the iRNA agent can be complementary to a human and more than one, e.g., two or three or more, non-human animals.
[0488] The methods described herein can be used to correlate any physiological effect of an iRNA agent on a human, e.g., any unwanted effect, such as a toxic effect, or any positive, or desired effect.
Delivery Module
[0489] In one aspect, the invention features a drug delivery conjugate or module, such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/454,265, filed on March 12, 2003, which is hereby incorporated by reference.
[0490] In addition, the invention includes iRNA agents described herein, e.g., a palindromic iRNA agent, an iRNA agent hving a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such a drug delivery conjugate or module.
[0491] The iRNA agents can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell or bone cell.
[0492] An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can be linked, e.g., coupled or bound, to the modular complex. The iRNA agent can interact with the condensing agent of the complex, and the complex can be used to deliver an iRNA agent to a cell, e.g., in vitro or in vivo. For example, the complex can be used to deliver an iRNA agent to a subject in need thereof, e.g., to deliver an iRNA agent to a subject having a disorder, e.g., a disorder described herein, such as a disease or disorder of the liver.
[0493] The fusogenic agent and the condensing agent can be different agents or the one and the same agent. For example, a polyamino chain, e.g., polyethyleneimine (PEI), can be the fusogenic and/or the condensing agent.
[0494] The delivery agent can be a modular complex. For example, the complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of):
(a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic interaction),
(b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane), and
(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl- galactos amine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic. Carrier agents
[0495] The carrier agent of a modular complex described herein can be a substrate for attachment of one or more of: a condensing agent, a fusogenic agent, and a targeting group. The carrier agent would preferably lack an endogenous enzymatic activity. The agent would preferably be a biological molecule, preferably a macromolecule. Polymeric biological carriers are preferred. It would also be preferred that the carrier molecule be biodegradable..
[0496] The carrier agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier molecule can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL),
poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L- lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Other useful carrier molecules can be identified by routine methods.
[0497] A carrier agent can be characterized by one or more of: (a) is at least 1 Da in size; (b) has at least 5 charged groups, preferably between 5 and 5000 charged groups; (c) is present in the complex at a ratio of at least 1 : 1 carrier agent to fusogenic agent; (d) is present in the complex at a ratio of at least 1 : 1 carrier agent to condensing agent; (e) is present in the complex at a ratio of at least 1 : 1 carrier agent to targeting agent.
Fusogenic agents
[0498] A fusogenic agent of a modular complex described herein can be an agent that is responsive to, e.g., changes charge depending on, the pH environment. Upon encountering the pH of an endosome, it can cause a physical change, e.g., a change in osmotic properties which disrupts or increases the permeability of the endosome membrane. Preferably, the fusogenic agent changes charge, e.g., becomes protonated, at pH lower than physiological range. For example, the fusogenic agent can become protonated at pH 4.5-6.5. The fusogenic agent can serve to release the iRNA agent into the cytoplasm of a cell after the complex is taken up, e.g., via endocytosis, by the cell, thereby increasing the cellular concentration of the iRNA agent in the cell.
[0499] In one embodiment, the fusogenic agent can have a moiety, e.g., an amino group, which, when exposed to a specified pH range, will undergo a change, e.g., in charge, e.g., protonation. The change in charge of the fusogenic agent can trigger a change, e.g., an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, the fusogenic agent, upon being exposed to the pH environment of an endosome, will cause a solubility or osmotic change substantial enough to increase the porosity of (preferably, to rupture) the endosomal membrane.
[0500] The fusogenic agent can be a polymer, preferably a polyamino chain, e.g., polyethyleneimine (PEI). The PEI can be linear, branched, synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, or lipid substituted PEI.
[0501] In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal. In some embodiment, the fusogenic agent can have an alpha helical structure. The fusogenic agent can be a membrane disruptive agent, e.g., mellittin.
[0502] A fusogenic agent can have one or more of the following characteristics: (a) is at least IDa in size; (b) has at least 10 charged groups, preferably between 10 and 5000 charged groups, more preferably between 50 and 1000 charged groups; (c) is present in the complex at a ratio of at least 1 : 1 fusogenic agent to carrier agent; (d) is present in the complex at a ratio of at least 1 : 1 fusogenic agent to condensing agent; (e) is present in the complex at a ratio of at least 1 : 1 fusogenic agent to targeting agent.
[0503] Other suitable fusogenic agents can be tested and identified by a skilled artisan. The ability of a compound to respond to, e.g., change charge depending on, the pH environment can be tested by routine methods, e.g., in a cellular assay. For example, a test compound is combined or contacted with a cell, and the cell is allowed to take up the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells and the endosome preparation compared to an endosome preparation from control cells. A change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cell and control cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in endosome population in the cells. The test compound can be labeled. In another type of assay, a modular complex described herein is constructed using one or more test or putative fusogenic agents. The modular complex can be constructed using a labeled nucleic acid instead of the iRNA. The ability of the fusogenic agent to respond to, e.g., change charge depending on, the pH environment, once the modular complex is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, as described above. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to, e.g., change charge depending on, the pH environment; and a second assay evaluates the ability of a modular complex that includes the test compound to respond to, e.g., change charge depending on, the pH environment.
Condensing agent
[0504] The condensing agent of a modular complex described herein can interact with (e.g., attracts, holds, or binds to) an iRNA agent and act to (a) condense, e.g., reduce the size or charge of the iRNA agent and/or (b) protect the iRNA agent, e.g., protect the iRNA agent against degradation. The condensing agent can include a moiety, e.g., a charged moiety, that can interact with a nucleic acid, e.g., an iRNA agent, e.g., by ionic interactions. The condensing agent would preferably be a charged polymer, e.g., a polycationic chain. The condensing agent can be a polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quarternary salt of a polyamine, or an alpha helical peptide.
[0505] A condensing agent can have the following characteristics: (a) at least IDa in size; (b) has at least 2 charged groups, preferably between 2 and 100 charged groups; (c) is present in the complex at a ratio of at least 1 : 1 condensing agent to carrier agent; (d) is present in the complex at a ratio of at least 1 : 1 condensing agent to fusogenic agent; (e) is present in the complex at a ratio of at least 1 : 1 condensing agent to targeting agent.
[0506] Other suitable condensing agents can be tested and identified by a skilled artisan, e.g., by evaluating the ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent. The ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent, e.g., to condense or protect the iRNA agent, can be evaluated by routine techniques. In one assay, a test agent is contacted with a nucleic acid, and the size and/or charge of the contacted nucleic acid is evaluated by a technique suitable to detect changes in molecular mass and/or charge. Such techniques include non-denaturing gel electrophoresis, immunological methods, e.g., immunoprecipitation, gel filtration, ionic interaction chromatography, and the like. A test agent is identified as a condensing agent if it changes the mass and/or charge (preferably both) of the contacted nucleic acid, compared to a control. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic cid; and a second assay evaluates the ability of a modular complex that includes the test compound to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid.
Amphipathic Delivery Agents
[0507] In one aspect, the invention features an amphipathic delivery conjugate or module, such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/455,050 (Attorney Docket No. 14174-065P01), filed on March 13, 2003, which is hereby incorporated by reference.
[0508] In addition, the invention include an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent hving a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such an amphipathic delivery conjugate.
[0509] An amphipathic molecule is a molecule having a hydrophobic and a hydrophilic region. Such molecules can interact with (e.g., penetrate or disrupt) lipids, e.g., a lipid bylayer of a cell. As such, they can serve as delivery agent for an associated (e.g., bound) iRNA (e.g., an iRNA or sRNA described herein). A preferred amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs descriebd herein) is a polymer. The polymer may have a secondary structure, e.g., a repeating secondary structure.
[0510] One example of an amphipathic polymer is an amphipathic polypeptide, e.g., a polypeptide having a secondary structure such that the polypeptide has a hydrophilic and a hybrophobic face. The design of amphipathic peptide structures (e.g., alpha-helical polypeptides) is routine to one of skill in the art. For example, the following references provide guidance: Grell et al. (2001) Protein design and folding: template trapping of self-assembled helical bundles J Pept Sci 7(3): 146-51 ; Chen et al. (2002) Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix J Pept Res 59(1): 18-33; Iwata et al. (1994) Design and synthesis of amphipathic 3(10)-helical peptides and their interactions with phospholipid bilayers and ion channel formation J Biol Chem 269(7):4928-33; Cornut et al. (1994) The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin FEBS Lett 349(l):29-33; Negrete et al. (1998) Deciphering the structural code for proteins: helical propensities in domain classes and statistical multiresidue information in alpha-helices. Protein Sci 7(6): 1368-79.
[0511] Another example of an amphipathic polymer is a polymer made up of two or more amphipathic subunits, e.g., two or more subunits containing cyclic moieties (e.g., a cyclic moiety having one or more hydrophilic groups and one or more hydrophobic groups). For example, the subunit may contain a steroid, e.g., cholic acid; or a aromatic moiety. Such moieties preferably can exhibit atropisomerism, such that they can form opposing hydrophobic and hydrophilic faces when in a polymer structure. [0512] The ability of a putative amphipathic molecule to interact with a lipid membrane, e.g., a cell membrane, can be tested by routine methods, e.g., in a cell free or cellular assay. For example, a test compound is combined or contacted with a synthetic lipid bilayer, a cellular membrane fraction, or a cell, and the test compound is evaluated for its ability to interact with, penetrate or disrupt the lipid bilayer, cell membrane or cell. The test compound can labeled in order to detect the interaction with the lipid bilayer, cell membrane or cell. In another type of assay, the test compound is linked to a reporter molecule or an iRNA agent (e.g., an iRNA or sRNA described herein) and the ability of the reporter molecule or iRNA agent to penetrate the lipid bilayer, cell membrane or cell is evaluated. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with a lipid bilayer, cell membrane or cell; and a second assay evaluates the ability of a construct (e.g., a construct described herein) that includes the test compound and a reporter or iRNA agent to interact with a lipid bilayer, cell membrane or cell.
[0513] An amphipathic polymer useful in the compositions described herein has at least 2, preferably at least 5, more preferably at least 10, 25, 50, 100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclic subunits). A single amphipathic polymer can be linked to one or more, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agents described herein). In some embodiments, an amphipathic polymer can contain both amino acid and cyclic subunits, e.g., aromatic subunits.
[0514] The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as "amphipathic iRNA constructs." Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.
[0515] In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.
[0516] The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.
[0517] The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.
[0518] The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an a-helical conformation as its secondary structure.
[0519] In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit
atropisomerism, e.g., a 2,2'-bis(substituted)-l-l '-binaphthyl or a 2,2'-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):
Figure imgf000214_0001
(M)
[0520] The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as "amphipathic iRNA constructs." Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell. [0521] In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.
[0522] The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.
[0523] The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.
[0524] The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an a-helical conformation as its secondary structure.
[0525] In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit
atropisomerism, e.g., a 2,2'-bis(substituted)-l-l '-binaphthyl or a 2,2'-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):
Figure imgf000216_0001
(M)
[0526] Referring to Formula M, Ri is C1-C100 alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; C1-C100 perfluoroalkyl; or OR5.
[0527] R2 is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR6; or Ci- C100 alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), S02, alkenyl, or alkynyl.
[0528] R3 is hydrogen, or when taken together with R4 froms a fused phenyl ring.
[0529] R4 is hydrogen, or when taken together with R3 froms a fused phenyl ring.
[0530] R5 is Ci-Cioo alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or C1-C100 perfluoroalkyl; and R6 is Ci- C100 alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), S02, alkenyl, or alkynyl.
Increasing cellular uptake of dsRNAs
[0531] A method of the invention that can include the administration of an iRNA agent and a drug that affects the uptake of the iRNA agent into the cell. The drug can be administered before, after, or at the same time that the iRNA agent is administered. The drug can be covalently linked to the iR A agent. The drug can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κΒ. The drug can have a transient effect on the cell.
[0532] The drug can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
[0533] The drug can also increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary drug's that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin- 1 beta, or gamma interferon.
Antisense molecules
[0534] In one embodiment of the present invention, antisense molecules or compounds are used as LDAs. The design, synthesis and use of compounds utilitzing RNaseH as a cleavage mechanisms are known in the art and described in "Antisense drug technology: principles, strategies, and applications" by Stanley T. Crooke, 2007, Marcel Dekker, New York.
Ribozymes
LDAs of the present invention may also be designed to be catalytic nucleic acids or ribozymes. The design, synthesis and use of ribozyme technology is known in the art and described in "Intracellular Ribozyme Applications: Principles and Protocols", by Rossi and Couture, 1999; Taylor & Francis, Inc. In one embodiment IncRNA transcript variants are designed to encode one or more ribozymes or ribozyme moieties. These ribozyme or ribozyme moieties are either active as encoded in the IncRNA transcript variant or when cleaved from the parent IncRNA transcript variant.
Aptamers
[0535] LDAs of the present invention may also be designed to be aptamers. The design, synthesis and use of aptamer technology is known in the art and described in "The Aptamer Handbook: Functional Oligonucleotides and Their Applications" by Klussman, 2006, Wiley VCH. In one embodiment, aptamers may be designed to target one or more features of a IncRNA transcript, transcript variant or IncRNA product.
Antibodies
[0536] In one embodiment of the present invention, antibodies and antibody technology may be employed to affect the expression or role of lncRNAs in a cell. As used herein, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
[0537] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
[0538] For the purposes herein, an "intact antibody" is one comprising heavy and light variable domains as well as an Fc region.
[0539] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V.sub.H) followed by a number of constant domains. Each light chain has a variable domain at one end (V.sub.L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
[0540] The term "variable domains" in reference to antibodies refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
[0541] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association..
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. [0542] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some
embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
[0543] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain VH connected to a light chain variable domain VL in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0544] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
[0545] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.
[0546] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
[0547] The term "hypervariable region" when used herein in reference to antibodies refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity determining region.
[0548] In one embodiment of the invention, antibodies which alter the levels or function of IncRNAs may be designed to directly or indirectly affect IncRNA targets. The preparation of antibodies, whether monoclonal or polyclonal, is know in the art. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999.
[0549] Antibodies of the present invention may be designed to directly affect the levels of IncRNA targets by binding directly to the IncRNA. Alternatively, antibodies can be designed to target the proteins encoded by the nearest neighbor genes of IncRNA, thereby altering the expression of the IncRNA.
Amino acid based LDAs
[0550] The LDAs of the present invention may also be polypeptide based molecules. These molecules may be "peptides," "polypeptides," or "proteins." While it is known in the art that these terms imply relative size, these terms as used herein should not be considered limiting with respect to the size of the various polypeptide based molecules referred to herein and which are encompassed within this invention.
[0551] The terms "amino acid" and "amino acids" refer to all naturally occurring L-alpha- amino acids. The amino acids are identified by either the one-letter or three-letter designations as follows: aspartic acid (Asp:D), isoleucine (Ile:I), threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q) methionine (Met:M), asparagines (Asn:N), where the amino acid is listed first followed parenthetically by the three and one letter codes, respectively.
[0552] The amino acid sequences of the LDAs of the invention may comprise naturally occurring amino acids and as such may be considered to be proteins, peptides, polypeptides, or fragments thereof. Alternatively, the LDAs may comprise both naturally and non-naturally occurring amino acids.
[0553] The term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences as compared to a native sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence. Ordinarily, variants will possess at least about 70% homology to a native sequence, and preferably, they will be at least about 80%, more preferably at least about 90% homologous to a native sequence.
[0554] "Homology" as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
[0555] By "homologs" as it applies to amino acid sequences is meant the corresponding sequence of other species having substantial identity to a second sequence of a second species.
[0556] "Analogs" is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain the properties of the parent polypeptide.
[0557] The term "derivative" is used synonymously with the term "variant" and refers to a molecule that has been modified or changed in any way relative to a reference molecule or starting molecule.
[0558] The present invention contemplates several types of LDAs which are amino acid based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives. As such, included within the scope of this invention are polypeptide based molecules containing substitutions, insertions and/or additions, deletions and covalently modifications. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the invention (e.g., at the N-terminal or C- terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.
[0559] "Substitutional variants" when referring to proteins are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. [0560] As used herein the term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
[0561] "Insertional variants" when referring to proteins are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. "Immediately adjacent" to an amino acid means connected to either the alpha- carboxy or alpha-amino functional group of the amino acid.
[0562] "Deletional variants" when referring to proteins are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
[0563] "Covalent derivatives" when referring to proteins include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and post- translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post- translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
[0564] Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post- translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the proteins used in accordance with the present invention. [0565] Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the .alpha. - amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).
[0566] Covalent derivatives specifically include fusion molecules in which proteins of the invention are covalently bonded to a non-proteinaceous polymer. The non-proteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymer not otherwise found in nature. However, polymers which exist in nature and are produced by recombinant or in vitro methods are useful, as are polymers which are isolated from nature. Hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularly useful are polyvinylalkylene ethers such a polyethylene glycol, polypropylene glycol. The proteins may be linked to various non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. No.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4, 179,337.
[0567] "Features" when referring to proteins are defined as distinct amino acid sequence- based components of a molecule. Features of the proteins of the present invention include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half- domains, sites, termini or any combination thereof.
[0568] As used herein when referring to proteins the term "surface manifestation" refers to a polypeptide based component of a protein appearing on an outermost surface.
[0569] As used herein when referring to proteins the term "local conformational shape" means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.
[0570] As used herein when referring to proteins the term "fold" means the resultant conformation of an amino acid sequence upon energy minimization. A fold may occur at the secondary or tertiary level of the folding process. Examples of secondary level folds include beta sheets and alpha helices. Examples of tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.
[0571] As used herein the term "turn" as it relates to protein conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
[0572] As used herein when referring to proteins the term "loop" refers to a structural feature of a peptide or polypeptide which reverses the direction of the backbone of a peptide or polypeptide and comprises four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997).
[0573] As used herein when referring to proteins the term "half-loop" refers to a portion of an identified loop having at least half the number of amino acid resides as the loop from which it is derived. It is understood that loops may not always contain an even number of amino acid residues. Therefore, in those cases where a loop contains or is identified to comprise an odd number of amino acids, a half-loop of the odd-numbered loop will comprise the whole number portion or next whole number portion of the loop (number of amino acids of the loop/2+/-0.5 amino acids). For example, a loop identified as a 7 amino acid loop could produce half-loops of 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 being 3 or 4).
[0574] As used herein when referring to proteins the term "domain" refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions.
[0575] As used herein when referring to proteins the term "half-domain" means portion of an identified domain having at least half the number of amino acid resides as the domain from which it is derived. It is understood that domains may not always contain an even number of amino acid residues. Therefore, in those cases where a domain contains or is identified to comprise an odd number of amino acids, a half-domain of the odd-numbered domain will comprise the whole number portion or next whole number portion of the domain (number of amino acids of the domain/2+/-0.5 amino acids). For example, a domain identified as a 7 amino acid domain could produce half-domains of 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 being 3 or 4). It is also understood that sub-domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half- domain or subdomain).
[0576] As used herein when referring to proteins the terms "site" as it pertains to amino acid based embodiments is used synonymous with "amino acid residue" and "amino acid side chain". A site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention.
[0577] As used herein the terms "termini or terminus" when referring to proteins refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions. The polypeptide based molecules of the present invention may be characterized as having both an N- terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C- termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.
[0578] Once any of the features have been identified or defined as a component of a molecule of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.
[0579] Modifications and manipulations can be accomplished by methods known in the art such as site directed mutagenesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
[0580] In one embodiment, artificial nucleosome proteins are provided. These proteins are only "artificial" in the sense that they are not identical to the wild-type proteins of the nucleosomes of the cells, or histones.
[0581] It is known in the art that nucleosomes undergo dynamic and necessary changes in order to facilitate presentation of genomic loci to the transcriptional machinery. This opening and closing of this chromosome packaging structure or "nucleosome respiration" is also
accompanied by changes in the histone proteins at the nucleosome core.
Taking advantage of this cellular process, the present invention provides compositions and methods for the manipulation and/or control of IncRNA gene expression and cellular regulation via the substitution or alteration of the protein components of nucleosomes.
[0582] More specifically, the present invention provides amino acid based compositions which replace one or more of the proteins contained in a nucleosome, but in doing so, alter the overall function of the nucleosome in a manner that alters the expression of one or more IncRNA genes. The IncRNA gene affected in this manner can be one whose DNA is packaged around the artificial nucleosome (e.g., the nucleosome harboring the protein variant) or one lying up or downstream of the artificial nucleosome whose transcription is altered as a result of the changes in the wild-type nucleosome. IncRNA genes have been localized via investigating chromatin state maps to sites in the genome having unique methylation patterns (Guttman, M., et al., Nature, 458, 223-227; 2009; Kalil, A., et al, Proc. Natl Acad. Set, 106, 1 1667-672; 2009). Given this unique location and the methylation patterns known to be associated with either up or down regulation of genes, the present invention provides methods of achieving directed gene expression at sites along chromosomes associated with increased rates of transcription.
[0583] In one embodiment are methods of altering the gene expression pattern of IncRNA genes within a cell comprising contacting the cells with artificial nucleosome proteins.
[0584] In one embodiment are methods of altering the epigenetic signature of a cells comprising contacting the cell with one or more artificial nucleosome proteins.
[0585] The artificial nucleosome proteins of the present invention include, but are not limited to analogs, homologs, variants or derivatives of wild-type histone proteins.
[0586] In one embodiment, the epigenetic signature, a component of which is the modificatoin pattern of the histones, e.g, methylation, may be changed by replacing wild type nucleosome proteins or histones with artificial histones engineered to have sites which are blocked such that cellular enzymes may no longer access or effect modifications to the proteins. These modifications may be permanent or transient. For example, blocking access of histones by histone methyl transferases, or other modifying enzymes, can alter the status of a cells in diseased states resulting in gene expression being "transcriptionally on" or "transcriptionally off. This embodiment is an attractive alternative to the small molecules being developed to modulate cellular enzymes which act to acetylate, methylate, ubiquitinate, etc, DNA or nucleosome components.
[0587] Other artificial proteins, not contained within the nucleosome, are also provided which may alter the expression of a IncRNA gene. These include variants or derivatives of nucleic acid binding proteins (e.g., RNA binding proteins, DNA binding proteins, chaperones, and the like) either localized in or which traffic to or within the nucleus.
Stem Cells
[0588] In one embodiment of the invention, IncRNA directed agents (LDAs) find utility in the study, research, manipulation, productionand application of stem cell-based technologies. According to the present invention, the differentiation status, survival, proliferation or regeneration of a stem cell or population of cells may be altered by the administration or introduction into a cell of a IncRNA directed agent (LDA). It is also contemplated that cellular populations may be affected or altered by the administration of a transcript which comprises a IncRNA transcript or gene. It is also contemplated that cells may be contacted with a combination of one or more LDAs or IncRNAs to modify the differentiation status, survival, proliferation or regeneration of the cells. [0589] Several protein coding genes have been identified in the art which affect the differentiation status, survival, proliferation or regeneration of stem cells or their precursors (See US Publication 20100166714 and 20060246446 which discloses genes affecting cardiac stem cells each of which is incorporated herein by reference in its entirety).
[0590] In one embodiment of the invention are provided compositions comprising IncRNA directed agents that function to regulate or modulate the expression of IncRNAs which in turn alter or modulate the protein coding genes which have been associated with differentiation status, survival, proliferation or regeneration of stem cells. For example, LDAs of the present invention may be used to target transcription factors known to alter the status of stem cell lineages. These LDAs amy target the transcripton factors at the DNA, R A or protein level but will always effect a change in the status or function of a IncRNA target.
III. Delivery of LP A
[0591] The delivery of a LDA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising a LDA, e.g. an iR A agent, dsR A, siRNA, antibody, etc to a subject.
Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the LDA. These alternatives are discussed further below.
[0592] "Introducing into a cell," when referring to a LDA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of a LDA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a LDA may also be "introduced into a cell," wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, LDAs can be injected into a tissue site or administered systemically. It is also contemplated by the inventors that introduction into cells or tissues may effected ex vivo, in situ and in ovo. In the case of transplants or within the field of stem cell technologies, it is contemplated that "introduction into a cell" will embrace the introduction to cells of any lineage or state, whether presently stem cells or which are intended to produce stem cells or progenitors or precursors thereof, as well as tissues, explants, organs and even organ systems.
Direct delivery
[0593] In general, any method of delivering a nucleic acid molecule can be adapted for use with a LDA (see e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5): 139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver a LDA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of a LDA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the LDA molecule to be administered.
Several studies have shown successful knockdown of gene products when an iRNA agent, one type of LDA, is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, ML, et al (2004) Retina 24: 132-138) and subretinal injections in mice (Reich, SL, et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al
(2005) Mol. Ther.l 1 :267-274) and can prolong survival of tumor-bearing mice (Kim, WL, et al
(2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, PL, et al (2002) BMC Neurosci. 3: 18; Shishkina, GT., et al (2004) Neuroscience 129:521-528; Thakker, ER., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101 : 17270-17275; Akaneya,Y., et al (2005) J.
Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, KA., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279: 10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering a LDA systemically for the treatment of a disease, the LDA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the molecule by endo- and exo-nucleases (in the case of nucleic acid based LDAs) in vivo. Modification of the RNA component of an LDA or the pharmaceutical carrier can also permit targeting of the LDA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA agent directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432: 173-178). Conjugation of a iRNA agent to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO., et al (2006) Nat. Biotechnol. 24: 1005-1015). In like fashion, the LDAs of the present invention may be conjugated to one or more aptamers. [0594] In an alternative embodiment, the LDA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system.
Positively charged cationic delivery systems facilitate binding of a LDA molecule (when negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of a LDA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to a LDA, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2): 107-116) that encases a LDA. The formation of vesicles or micelles further prevents degradation of the LDA when administered systemically. Methods for making and administering cationic-LDA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9: 1291-1300; Arnold, AS et al (2007) J. Hypertens. 25: 197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of LDAs (e.g., iRNAs) include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN., et al (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS., et al (2006) Nature 441 : 1 11-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26: 1087-1091), polyethyleneimine (Bonnet ME., et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3 :472-487), and polyamidoamines (Tomalia, DA., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16: 1799-1804). In some embodiments, a LDA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Patent No. 7, 427, 605, which is herein incorporated by reference in its entirety.
[0595] Methods of direct delivery are disclosed in PCT/US2007/079203 filed September 21, 2007 (Applicant docket number ALE-027) incorporated by reference herein in its entirety. These methods are useful in the present invention. Disclosed in PCT/US09/38437 filed 26-Mar-2009 (Applicant docket number ALN-062) and USSN 12/591,629 25-Nov-2009 (Applicant docket number ALN-080) are further methods of delivery useful in the present invention. Each of these documents is incorporated by reference herein in its entirety.
Vector encoded dsRNAs
[0596] In another aspect, LDAs (e.g., iRNAs, proteins or peptides) targeting the lncRNA can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al, TIG. (1996), 12:5-10; Skillern, A., et al, International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/221 14, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et ah, Proc. Natl. Acad. Sci. USA (1995) 92: 1292).
[0597] The individual strand or strands of a LDA (when RNA or DNA) can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
[0598] Expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of a LDA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of LDA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
[0599] LDA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit- TKO™). Multiple lipid transfections for LDA-mediated knockdowns targeting different regions of a IncRNA target over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
[0600] Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper- dependent or gutless adenovirus. Replication-defective viruses can also be advantageous.
Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors. Constructs for the recombinant expression of a LDA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the LDA in target cells. Other aspects to consider for vectors and constructs are further described below.
[0601] Vectors useful for the delivery of a LDA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the LDA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.
[0602] Expression of the LDA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-Dl -thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the transgene.
[0603] In a specific embodiment, viral vectors that contain nucleic acid sequences encoding a LDA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding a LDA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a cell, tissue or patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93 :644-651 (1994); Kiem et al., Blood 83 : 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3 : 1 10-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Patent Nos. 6, 143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference. [0604] In one embodiment, the LDAs of the present invention may be delivered via a bacterial delivery approach as disclosed in PCT Publication WO/2008/156702, the contents of which are incorporated herein in its entirety.
[0605] Adenoviruses are also contemplated for use in delivery of nucleic acid based LDAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3 :499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68: 143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91 :225-234 (1993); PCT Publication W094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing a LDA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
[0606] Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436, 146). In one embodiment, the LDA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61 : 3096-3101 ; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941 ; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
[0607] Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
[0608] The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
[0609] The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
IV. Formulations
Nucleic acid lipid particles
[0610] In one embodiment, a IncRNA LDA, e.g., a dsRNA, featured in the invention is fully encapsulated in a lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid- lipid particle. As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle, including SPLP. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA agent or a plasmid from which a iRNA agent is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos.
20060240093, 20070135372, and in International Application No. WO 2009082817. These applications are incorporated herein by reference in their entirety.
[0611] As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include "pSPLP," which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid- lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Nos. 5,976,567; 5,981,501 ; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964, each of which is incorporated herein by reference in its entirety. [0612] In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to LDA, e.g., dsRNA ratio) will be in the range of from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25: 1, from about 3 : 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1.
[0613] The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I -(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxy)propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane
(DLinDMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), l,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleyoxy-3-morpholinopropane (DLin- MA), l,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), 1 -Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-( - methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanedio (DOAP), l,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), l,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z, 12Z)-octadeca-9, 12- dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), l, l'-(2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethylazanediyl)didodecan-2-ol (Tech Gl), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
[0614] In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane is described in United States provisional patent application number 61/107,998 filed on October 23, 2008, which is herein incorporated by reference.
[0615] In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0 ± 20 nm and a 0.027 siRNA/Lipid Ratio. [0616] The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine
(DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
[0617] The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Q2), a PEG- dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Cie), or a PEG- distearyloxypropyl (C]s). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
[0618] In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
LNP01
[0619] In one embodiment, the lipidoid ND98-4HC1 (MW 1487) (see U.S. Patent Application No. 12/056,230, filed 3/26/2008, which is herein incorporated by reference), Cholesterol (Sigma- Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48: 10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
Figure imgf000236_0001
Formula 1
[0620] LNPOl formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
[0621] Additional exemplary lipid-dsRNA formulations are as follows:
Table 3 Lipid Nanoparticle formulations
Figure imgf000236_0002
Figure imgf000237_0001
Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine
DPPC : dipalmitoylphosphatidylcholine
PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)
PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-l,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)
[0622] SNALP (l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed April 15,
2009, which is hereby incorporated by reference in its entirety.
[0623] XTC comprising formulations are described, e.g., in U.S. Provisional Serial
No. 61/239,686, filed September 3, 2009 as well as PCT/US 10/22614 filed January 29, 2010 each of which is hereby incorporated by reference in its entirety. Further XTC formulations useful in the present invention are disclosed in PCT/US08/088588 filed 31 -Dec-2008 and
PCT/US08/88587 filed 31 -Dec-2008 and PCT/US09/041442 filed 22-Apr-2009 and
PCT/US09/061897 filed 23-Oct-2009 and PCT/US 10/38224 filed June 10, 2010, each of which is hereby incorporated by reference in its entirety.
[0624] MC3 comprising formulations are described, e.g., in U.S. Provisional Serial
No. 61/244,834, filed September 22, 2009, and U.S. Provisional Serial No. 61/185,800, filed
June 10, 2009, and PCT/US09/63933 filed November 10, 2009 and PCT/US09/63927 filed 10-
Nov-2009 and PCT/US09/63931 filed lO-Nov-2009 and PCT/US09/63897 filed lO-Nov-2009, each of which are hereby incorporated by reference in its entirety.
[0625] ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on November 10, 2009, which is hereby incorporated by reference in its entirety.
[0626] C12-200 comprising formulations are described in U.S. Provisional Serial No. 61/175,770, filed May 5, 2009, as well as PCT/USlO/33777 which are hereby incorporated by reference in its entirety.
[0627] Transfection reagents useful in the present invention are disclosed in US provisional 61/267,419 filed December 7, 2009, which is hereby incorporated by reference in its entirety.
[0628] Formulations for targeting immune cells useful in the present invention are disclosed in PCT/USlO/033747 filed May 5, 2010, which is hereby incorporated by reference in its entirety.
[0629] Pyrrolidine cationic lipids useful in the formulations of the present invention are disclosed in USSN 12/123,922 filed May 20, 2008 which is hereby incorporated by reference in its entirety. [0630] In one embodiment, the reagent that facilitates targeting construct uptake used herein comprises a cationic lipid as described in e.g., U.S. Application Ser. No. 61/267,419, filed 7 December 2009, and U.S. Application Ser. No. 61/334,398, filed 13 May 2010. In various embodiments, the composition described herein comprises a cationic lipid selected from the group consisting of: "Lipid H", "Lipid K"; "Lipid L", "Lipid M"; "Lipid P"; or "Lipid R", whose formulas are indicated as follows:
[
Figure imgf000239_0001
0637] Lipid R
[0638]
[0639] Also contemplated herein are various formulations of the lipids described above, such as, e.g., K8, P8 and L8 which refer to formulations comprising Lipid K, P, and L, respectively. Some exemplary lipid formulations for use with the methods and compositions described herein are found in e.g., Table 3B:
Table 3B. Example lipid formulations
Figure imgf000239_0002
[0640] In another embodiment, the composition described herein further comprises a lipid formulation comprising a lipid selected from the group consisting of Lipid H, Lipid K, Lipid L, Lipid M, Lipid P, and Lipid R, and further comprises a neutral lipid and a sterol. In particular embodiments, the lipid formulation comprises between approximately 25 mol % - 100 mol% of the lipid. In another embodiment, the lipid formulation comprises between 0 mol% - 50 mol% cholesterol. In still another embodiment, the lipid formulation comprises between 30 mol% - 65 mol% of a neutral lipid. In particular embodiments, the lipid formulation comprises the relative mol% of the components as listed in Table 3C as follows:
Table 3C. Example lipid formulae
Figure imgf000240_0001
Other particles
[0641] In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
[0642] In one embodiment, core-shell nanoparticles may be used for delivery to cells, tissues or organ systems. Such core-shell nanoparticles are described by Siegwart (Siegwart, et al., Combinatorial synthesis of chemically diverse core-shell nanoparticles for intracellular delivery, PNAS, PNAS Early edition, July 22, 2011 ; the contents of which are incorporated herein in their entirety) and comprise a cationic core to facilitate LDA complexation, with variation in the nature of the protonizable amine, and a shell with variation in polymer length and chemical properties. Block copolymers created by reacting epoxide
groups with amines and possessing poly(oligo(ethylene oxide) methacrylate) (POEOMA) with different lengths of the PEO side chain, may increase blood circulation time due to the PEO shell of the resulting nanoparticle. Anionic, cationic, zwitterionic, and hydrophobic blocks may also be used as shells. Liposomal formulations
[0643] There are many organized surfactant structures that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0644] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
[0645] In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
[0646] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
[0647] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
[0648] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side- effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin. [0649] Several reports have detailed the ability of liposomes to deliver agents including high- molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.
[0650] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et ah, Biochem. Biophys. Res. Commun, 1987, 147, 980-985).
[0651] Liposomes which are pH-sensitive or negatively charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et ah, Journal of Controlled Release, 1992, 19, 269-274).
[0652] One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
[0653] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et ah, Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et ah, Antiviral Research, 1992, 18, 259-265).
[0654] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al.
S.T.P.Pharma. Sci., 1994, 4, 6, 466).
[0655] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as
monosialoganglioside GMI, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al, FEBS Letters, 1987, 223, 42; Wu et al, Cancer Research, 1993, 53, 3765).
[0656] Various liposomes comprising one or more glycolipids are known in the art.
Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of
monosialoganglioside GMI, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al, disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GMI or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543, 152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
[0657] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2CI2ISG, that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 Bl and WO 90/04384 to Fisher. Liposome compositions containing 1- 20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No.
5,213,804 and European Patent No. EP 0 496 813 Bl). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
[0658] A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
[0659] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the
environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self- loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
[0660] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). [0661] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant.
Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxy ethylene surfactants are the most popular members of the nonionic surfactant class.
[0662] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
[0663] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
[0664] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
[0665] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0666] Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
[0667] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations of LDAs. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma. [0668] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
[0669] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Emulsions
[0670] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μιη in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et ah, in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 301). Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0671] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
[0672] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0673] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
[0674] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts,
benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
[0675] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
[0676] In one embodiment of the present invention, the compositions of LDAs are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
[0677] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously. [0678] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),
hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
[0679] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Patent Nos. 6, 191, 105; 7,063,860; 7,070,802; 7, 157,099; Constantinides et al, Pharmaceutical
Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral
administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Patent Nos. 6,191, 105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al, Pharmaceutical Research, 1994, 1 1, 1385; Ho et al, J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile LDA drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of nucleic acid based LDAs, e.g., iRNAs and nucleic acids, from the gastrointestinal tract, as well as improve the local cellular uptake. [0680] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the LDAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories-surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Penetration Enhancers
[0681] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of LDAs, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
[0682] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
[0683] Surfactants: In connection with the present invention, surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of LDAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
[0684] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1- monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1 -monocaprate, 1- dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, Ci-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al, J. Pharm. Pharmacol., 1992, 44, 651-654).
[0685] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw- Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783;
Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al, J. Pharm. Sci., 1990, 79, 579-583).
[0686] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of LDAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al, J. Control Rel., 1990, 14, 43-51).
[0687] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of LDAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal antiinflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al, J. Pharm. Pharmacol., 1987, 39, 621-626).
[0688] Agents that enhance uptake of LDAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al, PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA),
Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X- tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse,
Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France),
EcoTransfect (OZ Biosciences; Marseille, France), TransPassa Dl Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec™/LipoGen™ (Invivogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, CA, USA ), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B- Bridge International, Mountain View, CA, USA), among others.
[0689] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
Carriers
[0690] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al, DsRNA Res. Dev., 1995, 5, 1 15-121 ; Takakura et al, DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
Excipients
[0691] In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc). [0692] Pharmaceutically acceptable organic or inorganic excipients suitable for non- parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin,
hydroxymethylcellulose, polyvinylpyrrolidone and the like.
[0693] Formulations for topical administration of nucleic acids may include sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with LDAs which are nucleic acids can be used.
[0694] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
[0695] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible,
pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
[0696] Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
[0697] In addition to their administration, as discussed above, the LDAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by IncRNA expression. In any event, the administering physician can adjust the amount and timing of administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
[0698] Further, toxicity and therapeutic efficacy of compounds of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
[0699] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
V. Pharmaceutical compositions containing LDAs
a, iRNA
[0700] In one embodiment, the invention provides LDA pharmaceutical compositions containing a iRNA agent, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition is useful for treating a disease or disorder associated with the expression or activity of a IncRNA, such as pathological processes mediated by IncRNA expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.
[0701] The iRNA pharmaceutical compositions featured herein are administered in dosages sufficient to alter the expression of IncRNAs. In general, a suitable dose of iRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. The pharmaceutical composition may be administered once daily, or may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the LDA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the LDA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
[0702] The effect of a single dose on IncRNA levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
[0703] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
[0704] Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by IncRNA expression. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a transgene expressing a human IncRNA.
[0705] The present invention also includes pharmaceutical compositions and formulations that include the LDA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.
[0706] The LDA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
[0707] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
[0708] Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the LDAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,
dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
[0709] LDAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, LDAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, 1 -dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a Ci-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.
[0710] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral
formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and
ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. LDAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. LDAs complexing agents include poly-amino acids; polyimines; polyacrylates; poly alky lacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,
polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE- methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Patent 6,887,906, US Publn. No. 20030027780, and U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.
VI. Methods for treating diseases caused by expression of a IncRNA
[0711] The invention relates in particular to the use of a LDA targeting IncRNA and compositions containing at least one LDA, e.g., an iRNA agent, for the treatment of a IncRNA - mediated disorder or disease.
[0712] As used herein in the context of IncRNA expression, the terms "treat," "treatment," and the like, refer to relief from or alleviation of pathological processes mediated by IncRNA expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by IncRNA expression), the terms "treat," "treatment," and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition, such as slowing the progression of a malignancy or cancer, or increasing the clearance of an infectious organism to alleviate/reduce the symptoms caused by the infection, e.g., hepatitis caused by infection with a hepatitis virus.
[0713] By "lower" in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.
[0714] As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by IncRNA expression or an overt symptom of pathological processes mediated by IncRNA expression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by IncRNA expression, the patient's history and age, the stage of pathological processes mediated by IncRNA expression, and the administration of other agents that inhibit pathological processes mediated by IncRNA expression.
[0715] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a LDA and a pharmaceutically acceptable carrier. The pharmaceutical composition may also include a IncRNA transcript variant or product or feature thereof. As used herein, "pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount" refers to that amount of a LDA effective to produce the intended
pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter. For example, a therapeutically effective amount of a LDA targeting IncRNA can reduce IncRNA transcript levels by at least 10%.
[0716] The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.
[0717] For example, a composition containing a LDA targeting a IncRNA is used for treatment of a cancer. As used herein, cancer refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths. A cancer can be a tumor or hematological malignancy, and includes but is not limited to, all types of lymphomas/leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.
[0718] Leukemias, or cancers of the blood or bone marrow that are characterized by an abnormal proliferation of white blood cells i.e., leukocytes, can be divided into four major classifications including Acute lymphoblastic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Acute myelogenous leukemia or acute myeloid leukemia (AML) (AML with
translocations between chromosome 10 and 1 1 [t(10, 1 1)], chromosome 8 and 21 [t(8;21)], chromosome 15 and 17 [t(15; 17)], and inversions in chromosome 16 [inv(16)]; AML with multilineage dysplasia, which includes patients who have had a prior myelodysplasia syndrome (MDS) or myeloproliferative disease that transforms into AML; AML and myelodysplastic syndrome (MDS), therapy-related, which category includes patients who have had prior chemotherapy and/or radiation and subsequently develop AML or MDS; d) AML not otherwise categorized, which includes subtypes of AML that do not fall into the above categories; and e) Acute leukemias of ambiguous lineage, which occur when the leukemic cells can not be classified as either myeloid or lymphoid cells, or where both types of cells are present); and Chronic myelogenous leukemia (CML).
[0719] The types of carcinomas include, but are not limited to, papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sinonasal undifferentiated carcinoma.
[0720] The types of sarcomas include, but are not limited to, soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.
[0721] The invention further relates to the use of a LDA or a pharmaceutical composition thereof, e.g., for treating a cancer, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, the LDA or pharmaceutical composition thereof can also be administered in conjunction with one or more additional anti-cancer treatments, such as biological, chemotherapy and
radiotherapy. Accordingly, a treatment can include, for example, imatinib (Gleevac), all-trans- retinoic acid, a monoclonal antibody treatment (gemtuzumab, ozogamicin), chemotherapy (for example, chlorambucil, prednisone, prednisolone, vincristine, cytarabine, clofarabine, farnesyl transferase inhibitors, decitabine, inhibitors of MDR1), rituximab, interferon-a, anthracycline drugs (such as daunorubicin or idarubicin), L-asparaginase, doxorubicin, cyclophosphamide, doxorubicin, bleomycin, fludarabine, etoposide, pentostatin, or cladribine), bone marrow transplant, stem cell transplant, radiation thereapy, anti-metabolite drugs (methotrexate and 6- mercaptopurine), or any combination thereof.
[0722] Radiation therapy (also called radiotherapy, X-ray therapy, or irradiation) is the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered externally via external beam radiotherapy (EBRT) or internally via brachytherapy. The effects of radiation therapy are localised and confined to the region being treated. Radiation therapy may be used to treat almost every type of solid tumor, including cancers of the brain, breast, cervix, larynx, lung, pancreas, prostate, skin, stomach, uterus, or soft tissue sarcomas. Radiation is also used to treat leukemia and lymphoma.
[0723] Chemotherapy is the treatment of cancer with drugs that can destroy cancer cells. In current usage, the term "chemotherapy" usually refers to cytotoxic drugs which affect rapidly dividing cells in general, in contrast with targeted therapy. Chemotherapy drugs interfere with cell division in various possible ways, e.g. with the duplication of DNA or the separation of newly formed chromosomes. Most forms of chemotherapy target all rapidly dividing cells and are not specific to cancer cells, although some degree of specificity may come from the inability of many cancer cells to repair DNA damage, while normal cells generally can. Most
chemotherapy regimens are given in combination. Exemplary chemotherapeutic agents include , but are not limited to, 5-FU Enhancer, 9-AC, AG2037, AG3340, Aggrecanase Inhibitor, Aminoglutethimide, Amsacrine (m-AMSA), Asparaginase, Azacitidine, Batimastat (BB94), BAY 12-9566, BCH-4556, Bis-Naphtalimide, Busulfan, Capecitabine, Carboplatin,
Carmustaine+Polifepr Osan, cdk4/cdk2 inhibitors, Chlorombucil, CI-994, Cisplatin, Cladribine, CS-682, Cytarabine HCl, D2163, Dactinomycin, Daunorubicin HCl, DepoCyt, Dexifosamide, Docetaxel, Dolastain, Doxifluridine, Doxorubicin, DX8951f, E 7070, EGFR, Epirubicin, Erythropoietin, Estramustine phosphate sodium, Etoposide (VP 16-213), Farnesyl Transferase Inhibitor, FK 317, Flavopiridol, Floxuridine, Fludarabine, Fluorouracil (5-FU), Flutamide, Fragyline, Gemcitabine, Hexamethylmelamine (HMM), Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Interleukin-2, Irinotecan, ISI 641, Krestin, Lemonal DP 2202, Leuprolide acetate (LHRH-releasing factor analogue), Levamisole, LiGLA (lithium-gamma linolenate), Lodine Seeds, Lometexol, Lomustine (CCNU), Marimistat, Mechlorethamine HCl (nitrogen mustard), Megestrol acetate, Meglamine GLA, Mercaptopunne, Mesna, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Mitotane (o.p'-DDD), Mitoxantrone, Mitoxantrone HCl, MMI 270, MMP, MTA/LY 231514, Octreotide, ODN 698, OK-432, Oral Platinum, Oral Taxoid, Paclitaxel (TAXOL.RTM.), PARP Inhibitors, PD 183805, Pentostatin (2' deoxycoformycin), PKC 412, Plicamycin, Procarbazine HCl, PSC 833, Ralitrexed, RAS Farnesyl Transferase Inhibitor, RAS Oncogene Inhibitor, Semustine (methyl-CCNU), Streptozocin, Suramin, Tamoxifen citrate, Taxane Analog, Temozolomide, Teniposide (VM-26), Thioguanine, Thiotepa, Topotecan, Tyrosine Kinase, UFT
(Tegafur/Uracil), Valrubicin, Vinblastine sulfate, Vindesine sulfate, VX-710, VX-853, YM 116, ZD 0101, ZD 0473/Anormed, ZD 1839, ZD 9331.
[0724] Biological therapies use the body's immune system, either directly or indirectly, to fight cancer or to lessen the side effects that may be caused by some cancer treatments. In one sense, targeting IncRNA can be considered in this group of therapies in that it can stimulate immune system action against a tumor, for example. However, this approach can also be considered with other such biological approaches, e.g., immune response modifying therapies such as the administration of interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents are also envisioned as anti-cancer therapies to be combined with the inhibition of IncRNA . Small molecule targeted therapy drugs are generally inhibitors of enzymatic domains on mutated, overexpressed, or otherwise critical proteins within the cancer cell, such as tyrosine kinase inhibitors imatinib (Gleevec/Glivec) and gefitinib (Iressa). Examples of monoclonal antibody therapies that can be used with a LDA or pharmaceutical composition thereof include, but are not limited to, the anti-HER2/neu antibody trastuzumab (Herceptin) used in breast cancer, and the anti-CD20 antibody rituximab, used in a variety of B-cell malignancies. The growth of some cancers can be inhibited by providing or blocking certain hormones. Common examples of hormone-sensitive tumors include certain types of breast and prostate cancers. Removing or blocking estrogen or testosterone is often an important additional treatment. In certain cancers, administration of hormone agonists, such as progestogens may be therapeutically beneficial.
[0725] Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the patient's own immune system to fight the tumor, and include, but are not limited to, intravesical BCG immunotherapy for superficial bladder cancer, vaccines to generate specific immune responses, such as for malignant melanoma and renal cell carcinoma, and the use of Sipuleucel-T for prostate cancer, in which dendritic cells from the patient are loaded with prostatic acid phosphatase peptides to induce a specific immune response against prostate- derived cells.
[0726] In some embodiments, a LDA targeting lncRNA is administered in combination with an angiogenesis inhibitor. In some embodiments, the angiogenesis inhibitors for use in the methods described herein include, but are not limited to, monoclonal antibody therapies directed against specific pro-angiogenic growth factors and/or their receptors. Examples of these are: bevacizumab (Avastin®), cetuximab (Erbitux®), panitumumab (Vectibix™), and trastuzumab (Herceptin®). In some embodiments, the angiogenesis inhibitors for use in the methods described herein include but are not limited to small molecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor receptors. The three TKIs that are currently approved as anti-cancer therapies are erlotinib (Tarceva®), sorafenib (Nexavar®), and sunitinib (Sutent®). In some embodiments, the angiogenesis inhibitors for use in the methods described herein include but are not limited to inhibitors of mTOR (mammalian target of rapamycin) such as temsirolimus (Toricel™), bortezomib (Velcade®), thalidomide (Thalomid®), and Doxycyclin,
[0727] In other embodiments, the angiogenesis inhibitors for use in the methods described herein include one or more drugs that target the VEGF pathway, including, but not limited to, Bevacizumab (Avastin®), sunitinib (Sutent®), and sorafenib (Nexavar®). Additional VEGF inhibitors include CP-547,632 (3-(4-Bromo-2,6-difluoro- benzyloxy)-5-[3-(4-pyrrolidin 1-yl- butyl)-ureido]-isothiazole-4- carboxylic acid amide hydrochloride; Pfizer Inc. , NY), AG13736, AG28262 (Pfizer Inc.), SU5416, SU11248, & SU6668 (formerly Sugen Inc., now Pfizer, New York, New York), ZD-6474 (AstraZeneca), ZD4190 which inhibits VEGF-R2 and -Rl
(AstraZeneca), CEP-7055 (Cephalon Inc., Frazer, PA), PKC 412 (Novartis), AEE788 (Novartis), AZD-2171), NEXAVAR® (BAY 43-9006, sorafenib; Bayer Pharmaceuticals and Onyx
Pharmaceuticals), vatalanib (also known as PTK-787, ZK-222584: Novartis & Schering: AG), MACUGEN® (pegaptanib octasodium, NX- 1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862 (glufanide disodium, Cytran Inc. of Kirkland, Washington, USA), VEGFR2-selective monoclonal antibody DC 101 (ImClone Systems, Inc.), angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron (Emeryville, California), Sirna-027 (an siR A-based VEGFR1 inhibitor, Sirna Therapeutics, San Francisco, CA) Caplostatin, soluble ectodomains of the VEGF receptors, Neovastat (Sterna Zentaris Inc; Quebec City, CA), ZM323881
(CalBiochem. CA, USA), pegaptanib (Macugen) (Eyetech Pharmaceuticals), an anti-VEGF aptamer and combinations thereof.
[0728] In other embodiments, the angiogenesis inhibitors for use in the methods described herein include anti-angiogenic factors such as alpha-2 antiplasmin (fragment), angiostatin (plasminogen fragment), antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59 complement fragment, endostatin (collagen XVIII fragment), fibronectin fragment, gro- beta ( a C-X-C chemokine), heparinases heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, interferon inducible protein (IP- 10), interleukin-12, kringle 5 (plasminogen fragment), beta-thromboglobulin, EGF (fragment), VEGF inhibitor, endostatin, fibronection (45 kD fragment), high molecular weight kininogen (domain 5), NK1, NK2, NK3 fragments of HGF, PF-4, serpin proteinase inhibitor 8, TGF-beta-1, thrombospondin-1, prosaposin, p53, angioarrestin, metalloproteinase inhibitors (TIMPs), 2- Methoxyestradiol, placental ribonuclease inhibitor, plasminogen activator inhibitor, prolactin 16kD fragment, proliferin-related protein (PRP), retinoids, tetrahydrocortisol-S transforming growth factor-beta (TGF-b), vasculostatin, and vasostatin (calreticulin fragment).pamidronate thalidomide, TNP470, the bisphosphonate family such as amino-bisphosphonate zoledronic acid, bombesin/gastrin-releasing peptide (GRP) antagonists such as RC-3095 and RC-3940-II (Bajol AM, et. al., British Journal of Cancer (2004) 90, 245-252), anti-VEGF peptide RRKRRR (dRK6) (Seung-Ah Yoo, J.Immuno, 2005, 174: 5846-5855).
[0729] Efficacy of treatment or amelioration of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of a LDA targeting IncRNA or pharmaceutical composition thereof, "effective against" a cancer indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of cancer. [0730] A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given LDA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.
[0731] The invention relates in particular to the use of a LDA targeting IncRNA and compositions containing at least one such LDA for the treatment of a IncRNA -mediated disorder or disease. For example, a composition containing a LDA targeting a IncRNA is used for treatment of an infectious disease or disorder, for example, in a subject having an infection. In some preferred embodiments the subject has an infection or is at risk of having an infection. An "infection" as used herein refers to a disease or condition attributable to the presence in a host of a foreign organism or agent that reproduces within the host. Infections typically involve breach of a normal mucosal or other tissue barrier by an infectious organism or agent. A subject that has an infection is a subject having objectively measurable infectious organisms or agents present in the subject's body. A subject at risk of having an infection is a subject that is predisposed to develop an infection. Such a subject can include, for example, a subject with a known or suspected exposure to an infectious organism or agent. A subject at risk of having an infection also can include a subject with a condition associated with impaired ability to mount an immune response to an infectious organism or agent, e.g., a subject with a congenital or acquired immunodeficiency, a subject undergoing radiation therapy or chemotherapy, a subject with a burn injury, a subject with a traumatic injury, a subject undergoing surgery or other invasive medical or dental procedure.
[0732] Infections are broadly classified as bacterial, viral, fungal, or parasitic based on the category of infectious organism or agent involved. Other less common types of infection are also known in the art, including, e.g., infections involving rickettsiae, mycoplasmas, and agents causing scrapie, bovine spongiform encephalopthy (BSE), and prion diseases (e.g., kuru and Creutzfeldt- Jacob disease). Examples of bacteria, viruses, fungi, and parasites which cause infection are well known in the art. An infection can be acute, subacute, chronic, or latent, and it can be localized or systemic. As defined herein, a "chronic infection" refers to those infections that are not cleared by the normal actions of the innate or adaptive immune responses and persist in the subject for a long duration of time, on the order of weeks, months, and years. A chronic infection may reflect latency of the infectious agent, and may be include periods in which no infectious symptoms are present, i.e., asymptomatic periods. Examples of chronic infections include, but are not limited to, HIV infection and herpesvirus infections. Furthermore, an infection can be predominantly intracellular or extracellular during at least one phase of the infectious organism's or agent's life cycle in the host.
[0733] Exemplary viruses include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III), HIV-2, LAV or HTLV-III/LAV, or HIV-III, and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); adenovirus; Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses, i.e., Rotavirus A, Rotavirus B. Rotavirus C); Birnaviridae; Hepadnaviridae (Hepatitis A and B viruses); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, Human herpes virus 6, Human herpes virus 7, Human herpes virus 8, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Epstein-Barr virus; Rous sarcoma virus; West Nile virus; Japanese equine encephalitis, Norwalk, papilloma virus, parvovirus B19;
Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); Hepatitis D virus, Hepatitis E virus, and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=enterally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and
astroviruses).
[0734] Bacteria include both Gram negative and Gram positive bacteria. Examples of Gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species. Examples of Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M. intracellular e, M.
kansasii, M. gordonae, M. leprae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group),
Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic spp.), Streptococcus pneumoniae, pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae (Hemophilus influenza B, and Hemophilus influenza non-typable) , Bacillus anthracis,
Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia, Actinomyces israelii, meningococcus, pertussis, pneumococcus, shigella, tetanus, Vibrio cholerae, yersinia,
Pseudomonas species, Clostridia species, Salmonella typhi, Shigella dysenteriae, Yersinia pestis, Brucella species, Legionella pneumophila, Rickettsiae, Chlamydia, Clostridium perfringens, Clostridium botulinum, Staphylococcus aureus, Pseudomonas aeruginosa, Cryptosporidium parvum, Streptococcus pneumoniae, and Bordetella pertussis.
[0735] Exemplary fungi and yeast include, but are not limited to, Cryptococcus neoformans, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis , Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Aspergillus fumigatus, Aspergillus flavus, Blastomyces dermatitidis , Aspergillus clavatus, Cryptococcus neoformans, Chlamydia trachomatis, Coccidioides immitis, Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii, Nocardia spp, Histoplasma capsulatum, Pneumocystis jirovecii (or Pneumocystis carinii), Stachybotrys chartarum, and any combination thereof.
[0736] Exemplary parasites include, but are not limited to: Entamoeba histolytica;
Plasmodium species (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax), Leishmania species (Leishmania tropica, Leishmania braziliensis,
Leishmania donovani), Toxoplasmosis (Toxoplasma gondii), Trypanosoma gambiense,
Trypanosoma rhodesiense (African sleeping sickness), Trypanosoma cruzi (Chagas' disease), Helminths (flat worms, round worms), Babesia microti, Babesia divergens, Giardia lamblia, and any combination thereof.
[0737] The invention further relates to the use of a LDA targeting IncRNA and compositions containing at least one such LDA for the treatment of an infectious disease, such as hepatitis B or a chronic bacterial infection, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating such infectious diseases or disorders (e.g., antibiotics, anti-viral agents). For example, in certain embodiments, administration of a dsRNA targeting IncRNA is administered in combination with an antibacterial agent. Examples of anti-bacterial agents useful for the methods described herein include, but are not limited to, natural penicillins, semi-synthetic penicillins, clavulanic acid, cephalosporins, bacitracin, ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, piperacillin, methicillin, dicloxacillin, nafcillin, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor, cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone, ceftazidine, moxalactam, carbapenems, imipenems, monobactems, euztreonam, vancomycin, polymyxin, amphotericin B, nystatin, imidazoles, clotrimazole, miconazole, ketoconazole, itraconazole, fluconazole, rifampins, ethambutol, tetracyclines, chloramphenicol, macrolides,
aminoglycosides, streptomycin, kanamycin, tobramycin, amikacin, gentamicin, tetracycline, minocycline, doxycycline, chlortetracycline, erythromycin, roxithromycin, clarithromycin, oleandomycin, azithromycin, chloramphenicol, quinolones, co-trimoxazole, norfloxacin, ciprofloxacin, enoxacin, nalidixic acid, temafloxacin, sulfonamides, gantrisin, and trimethoprim; Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin;
Azithromycin; Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride; Bacitracin;
Bacitracin Methylene Disalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium;
Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; Biphenamine Hydrochloride;
Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate; Capreomycin Sulfate; Carbadox;
Carbenicillin Disodium; Carbenicillin Indanyl Sodium; Carbenicillin Phenyl Sodium;
Carbenicillin Potassium; Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate; Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride; Cefetecol; Cefixime; Cefinenoxime Hydrochloride; Cefinetazole; Cefinetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan;
Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole;
Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium; Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine; Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;
Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; Cephalexin Hydrochloride;
Cephaloglycin; Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine;
Cetocycline Hydrochloride; Cetophenicol; Chloramphenicol; Chloramphenicol Palmitate;
Chloramphenicol Pantothenate Complex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate; Chloroxylenol; Chlortetracycline Bisulfate; Chlortetracycline Hydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride; Cirolemycin; Clarithromycin;
Clinafloxacin Hydrochloride; Clindamycin; Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride; Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; Cloxacillin Sodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin; Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone; Daptomycin; Demeclocycline;
Demeclocycline Hydrochloride; Demecycline; Denofungin; Diaveridine; Dicloxacillin;
Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline; Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; Droxacin Sodium;
Enoxacin; Epicillin; Epitetracycline Hydrochloride; Erythromycin; Erythromycin Acistrate; Erythromycin Estolate; Erythromycin Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Lactobionate; Erythromycin Propionate; Erythromycin Stearate; Ethambutol Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine; Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin; Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid; Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin; Hetacillin Potassium; Hexedine; Ibafloxacin; Inipenem; Isoconazole; Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin; Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;
Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; Meclocycline Subsalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem; Methacycline; Methacycline Hydrochloride; Methenamine; Methenamine Hippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim; Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin Hydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; Nalidixate Sodium; Nalidixic Acid;
Natamycin; Nebramycin; Neomycin Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate; Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone; Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium; Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin; Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin G Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V; Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V Potassium; Pentizidone Sodium; Phenyl Aminosalicylate; Piperacillin Sodium; Pirbenicillin Sodium; Piridicillin Sodium; Pirlimycin Hydrochloride; Pivampicillin Hydrochloride;
Pivampicillin Pamoate; Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin;
Propikacin; Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin;
Rosaramicin Butyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate; Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium;
Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin; Sisomicin Sulfate; Sparfloxacin;
Spectinomycin Hydrochloride; Spiramycin; Stallimycin Hydrochloride; Steffimycin;
Streptomycin Sulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide; Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium; Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine; Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine;
Sulfamoxole; Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole; Sulfathiazole;
Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine; Sulfomyxin;
Sulopenem; Sultamicillin; Suncillin Sodium; Talampicillin Hydrochloride; Teicoplanin;
Temafloxacin Hydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;
Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol; Thiphencillin Potassium;
Ticarcillin Cresyl Sodium; Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin;
Vancomycin Hydrochloride; Virginiamycin; and Zorbamycin.
[0738] In other embodiments, administration of a LDA targeting IncRNA is performed in combination with an anti-viral medicament or agent. Exemplary antiviral agents useful for the methods described herein include, but are not limited to, immunoglobulins, amantadine, interferon, nucleoside analogues, and protease inhibitors. Specific examples of antiviral agents include but are not limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate;
Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate;
Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscamet Sodium; Fosfonet Sodium;
Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; Somantadine Hydrochloride; Sorivudine; Statolon;
Stavudine; Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride; Vidarabine;
Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine; Zidovudine; and Zinviroxime.
[0739] In other embodiments, administration of a LDA targeting IncRNA is performed in combination with an anti-fungal medicament or agent. An "antifungal medicament" is an agent that kills or inhibits the growth or function of infective fungi. Anti-fungal medicaments are sometimes classified by their mechanism of action. Some anti-fungal agents function as cell wall inhibitors by inhibiting glucose synthase, other antifungal agents function by destabilizing membrane integrity, and other antifungal agents function by breaking down chitin (e.g., chitinase) or immunosuppression (501 cream). Thus, exemplary antifungal medicaments useful for the methods described herein include, but are not limited to, imidazoles, 501 cream, and Acrisorcin, Ambruticin, Amorolfine, Amphotericin B, Azaconazole, Azaserine, Basifungin, BAY 38-9502, Bifonazole, Biphenamine Hydrochloride, Bispyrithione Magsulfex, Butenafine, Butoconazole Nitrate, Calcium Undecylenate, Candicidin, Carbol-Fuchsin, Chitinase,
Chlordantoin, Ciclopirox, Ciclopirox Olamine, Cilofungin, Cisconazole, Clotrimazole,
Cuprimyxin, Denofungin, Dipyrithione, Doconazole, Econazole, Econazole Nitrate,
Enilconazole, Ethonam Nitrate, Fenticonazole Nitrate, Filipin, FK 463, Fluconazole, Flucytosine, Fungimycin, Griseofulvin, Hamycin, Isoconazole, Itraconazole, Kalafungin, Ketoconazole, Lomofungin, Lydimycin, Mepartricin, Miconazole, Miconazole Nitrate, MK 991, Monensin, Monensin Sodium, Naftifine Hydrochloride, Neomycin Undecylenate, Nifuratel, Nifurmerone, Nitralamine Hydrochloride, Nystatin, Octanoic Acid, Orconazole Nitrate, Oxiconazole Nitrate, Oxifungin Hydrochloride, Parconazole Hydrochloride, Partricin, Potassium Iodide, Pradimicin, Proclonol, Pyrithione Zinc, Pyrrolnitrin, Rutamycin, Sanguinarium Chloride, Saperconazole, Scopafungin, Selenium Sulfide, Sertaconazole, Sinefungin, Sulconazole Nitrate, Terbinafine, Terconazole, Thiram, Ticlatone, Tioconazole, Tolciclate, Tolindate, Tolnaftate, Triacetin, Triafungin, UK 292, Undecylenic Acid, Viridofulvin, Voriconazole, Zinc Undecylenate, and Zinoconazole Hydrochloride.
[0740] In further embodiments, administration of a LDA targeting IncRNA is administered in combination with an anti-parasitic medicament or agent. An "antiparasitic medicament" refers to an agent that kills or inhibits the growth or function of infective parasites. Examples of antiparasitic medicaments, also referred to as parasiticides, useful for the methods described herein include, but are not limited to,albendazole, amphotericin B, benznidazole, bithionol, chloroquine HC1, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, doxycycline, eflomithine, furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine, quinacrine HC1, quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium (sodium antimony gluconate), suramin, tetracycline, thiabendazole, timidazole, trimethroprim-sulfamethoxazole, and tryparsamide, some of which are used alone or in combination with others.
[0741] The LDA and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.
[0742] Patients can be administered a therapeutic amount of an LDA, such as 0.5 mg/kg, 1.0 mg kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The LDA can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the LDA can reduce IncRNA transcript levels, e.g., in a cell, tissue, blood, urine or other compartment of a patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
[0743] Before administration of a full dose of the LDA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.
[0744] Genetic predisposition plays a role in the development of some cancers and hematological malignancies. Therefore, a patient in need of a IncRNA LDA may be identified by taking a family history, or, for example, screening for one or more genetic markers or variants. A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a IncRNA LDA. For example, certain variants in the BRCAl and BRCA2 genes are known to cause an increased risk for breast and ovarian cancers. A DNA test may also be performed on the patient to identify a mutation in the IncRNA, before a IncRNA LDA is administered to the patient.
[0745] Owing to the inhibitory effects on IncRNA expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.
VII. Kits
[0746] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, reagents for generating LDAs, including iRNA agents and specifically siRNA molecules are included in a kit. The kit may further include reagents or instructions for creating or synthesizing the LDA. It may also include one or more buffers, such as a nuclease buffer, transcription buffer, or a hybridization buffer, compounds for preparing the DNA template or a dsRNA, and components for isolating the resultant template, dsRNA, or siRNA. Other kits of the invention may include components for making a nucleic acid array comprising iRNA agents, e.g., siRNA, and thus, may include, for example, a solid support.
[0747] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the LDAs, e.g., nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
[0748] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. In some embodiments, labeling dyes are provided as a dried power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrograms or at least or at most those amounts of dried dye are provided in kits of the invention. The dye may then be resuspended in any suitable solvent, such as DMSO.
[0749] The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the LDA, e.g., nucleic acid formulations are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
[0750] The kits of the present invention may also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
[0751] Kits may also include components that facilitate isolation of a DNA template, long dsRNA, or iRNA agent, e.g., siRNA. It may also include components that preserve or maintain the nucleic acids or that protect against their degradation. Such components may be RNAse-free or protect against RNAses, such as RNase inhibitors. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.
[0752] A kit can include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be
implemented.
VIII. Bioprocessing
[0753] In one embodiment of the invention are methods for producing a biological product in a host cell by contacting the cell with an iRNA agent capable of modulating expression of a target gene, wherein the modulation enhances production of the biological product. According to the present invention, bioprocessing methods may be improved by targeting (using one or more of the LDAs of the present invention) IncRNA genes or transcripts expressed endogenously or expressed as a result of engineering the host cells to express said targets. They may also be improved by supplementing, replacing or adding one or more IncRNA transcripts or transcript variants or IncRNA products.
[0754] Bioprocessing methods of using RNA effector molecules (e.g., siRNA, miRNA, dsRNA, saRNA, shRNA, piRNA, tkRNAi, eiRNA, pdRNA, a gapmer, an antagomir, or a ribozyme) are disclosed in co-owned applications 61/223,370, filed July 6, 2009, entitled COMPOSITIONS AND METHODS FOR ENHANCING PRODUCTION OF A BIOLOGICAL PRODUCT by Maraganore et al.; U.S. Provisional Patent Application No. 61/244,868 filed September 22, 2009, entitled COMPOSITIONS AND METHODS FOR ENHANCING
PRODUCTION OF A BIOLOGICAL PRODUCT, by Maraganore et al.; U.S. Provisional Patent Application No. 61/293,980, filed January 1 1, 2010, entitled COMPOSITIONS AND
METHODS FOR ENHANCING PRODUCTION OF A BIOLOGICAL PRODUCT, by
Rossomando et al.; U.S. Provisional Patent Application No. 61/319,589, filed March 31, 2010, entitled CELL-BASED BIOPROCESSING by Rossomando et al.; and U.S. Provisional Patent Application No. 61/354,932, filed June 15, 2010, entitled CHINESE HAMSTER OVARY (CHO) CELL TRANSCRIPTOME, CORRESPONDING SIRNAS AND USES THEREOF, by Rossomando et al.; each of which is incorporated fully herein by reference. These methods may be employed in the process of targeting lncRNAs to improve bioprocessing. EXAMPLES
Example 1. Nearest Neighbor Analysis of IncRNA genes and transcripts
[0755] For each ENST transcript, the genomic positions of exons were parsed from the "Homo_sapiens. GRCh37.58.gif ' annotation file in the Ensembl database, and saved according to ENST identifier, exon number, and position. For each IncRNA, the neighboring exons (and thus ENST transcripts) were selected on the criteria of minimal distance between that exon and the IncRNA, one exon in each of the "left" (upstream) and "right" (downstream) directions. In the event of multiple ENST transcripts sharing the same IncRNA-neighbor exon, the ENST transcript listed closer to the exon from the "Homo_sapiens. GRCh37.58.gif ' table was chosen. ENST transcripts were then mapped back to encoding ENSG genes using the
"Homo_sapiens. GRCh37.58.gif ' table. The results of this analysis are presented in Table 2.
Example 2. siRNA Design
[0756] Using the IncRNA transcripts identified in Example 1, oligonucleotide design was carried out to identify siRNAs targeting the IncRNA of Table 2. All sequences were obtained as described above using both the ENSEMBL and NCBI datasets publicly available.
All siRNA duplexes were designed with 100% identity to their respective transcripts with a total of 372, 148 sense and antisense oligonucleotides were designed. These are presented in in the sequence listing and as lengthy Table 1 submitted with the present application and incorporated herein in its entirety.
siRNA Design and Specificity Prediction
[0757] The specificity of the 19mer oligonucleotide set was predicted from each sequence. The IncRNA were used in a comprehensive search against their respective human, or mouse and rat transcriptomes (defined as the set of ENSG_ and ENST_ records within the ENSEMBL and NCBI Refseq datasets) using the BLASTN algorithm and a perl script was used to parse the alignment and generate a scorescore based on the position and number of mismatches between the siRNA and any potential Off-target' transcript. The off-target score is weighted to emphasize differences in the 'seed' region of siRNAs, in positions 2-9 from the 5' end of the molecule. The off-target score is calculated as follows: mismatches between the oligo and the transcript are given penalties. A mismatch in the seed region in positions 2-9 of the oligo is given a penalty of 2.8; mismatches in the putative cleavage sites 10 and 11 are given a penalty of 1.2, and all other mismatches a penalty of 1. The off-target score for each oligo-transcript pair is then calculated by summing the mismatch penalties. The lowest off-target score from all the oligo-transcript pairs is then determined and used in subsequent sorting of oligos. Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderate specific. In picking which oligonucleotides to synthesize, these scores can be sorted from high to low by the off-target score of the antisense strand and the best (lowest off-target score) oligonucleotide pairs may be synthesized for further study.
Example 3. Oligonucleotide synthesis
Source of reagents
[0758] Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
Oligonucleotide Synthesis.
[0759] All oligonucleotides are synthesized on an AKTAoligopilot synthesizer.
Commercially available controlled pore glass solid support (dT-CPG, 500A, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5'-0-dimethoxytrityl N6-benzoyl- 2'-?-butyldimethylsilyl-adenosine-3 '-0-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-0- dimethoxytrityl-N4-acetyl-2'-?-butyldimethylsilyl-cytidine-3'-0-N,N'-diisopropyl-2- cyanoethylphosphoramidite, 5 ' -0-dimethoxytrityl-N2— isobutryl-2 ' -?-butyldimethylsilyl- guanosine-3 '-0-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and 5'-0-dimethoxytrityl-2'-/- butyldimethylsilyl-uridine-3 '-0-N,N'-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2'-F phosphoramidites, 5'- 0-dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3'-0-N,N'-diisopropyl-2-cyanoethyl- phosphoramidite and 5'-0-dimethoxytrityl-2'-fluro-uridine-3 '-0-N,N'-diisopropyl-2- cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH3CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO- oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6- lutidine/ACN (1 : 1 v/v) is used.
[0760] 3'-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans- 4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5 '-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5 '-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH3CN in the presence of 5-(ethylthio)-lH-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert- butyl hydroperoxide/acetonitrile/water (10: 87: 3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to
phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM
Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.
Deprotection I (Nucleobase Deprotection)
[0761] After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3 : 1)] for 6.5 h at 55°C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2 x 40 mL portions of ethanol/water (1 : 1 v/v). The volume of the mixture is then reduced to ~ 30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.
Deprotection II (Removal of 2'-TBDMS group)
[0762] The dried residue is resuspended in 26 mL of triethylamine, triethylamine
trihydrofiuoride (TEA»3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60°C for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5.
Oligonucleotide is stored in a freezer until purification.
Analysis
[0763] The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.
HPLC Purification
[0764] The ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH3CN, 1M NaBr (buffer B).
Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotidess are diluted in water to 150 μΐ, and then pipetted into special vials for CGE and LC/MS analysis. Compounds are then analyzed by LC- ESMS and CGE.
iRNA preparation
[0765] For the general preparation of iRNA agents, specifically siRNAs, equimolar amounts of sense and antisense strand are heated in IxPBS at 95°C for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 4.
Table 4: Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an
Figure imgf000278_0001
Synthesis of Sequences
[0766] Sequences are synthesized on a MerMade 192 synthesizer at Ι μηιοΐ scale.
[0767] For all the sequences in the list, 'endolight' chemistry may be applied as detailed below.
All pyrimidines (cytosine and uridine) in the sense strand contain 2'-0-Methyl bases (2' O-Methyl C and 2'-0-Methyl U)
In the antisense strand, pyrimidines adjacent to (towards 5' position) ribo A nucleoside are replaced with their corresponding 2-O-Methyl nucleosides A two base dTsdT extension at 3' end of both sense and antisense sequences are introduced
The sequence file is converted to a text file to make it compatible for loading in the MerMade 192 synthesis software
Synthesis, Cleavage and deprotection: [0768] The synthesis of sequences uses solid supported oligonucleotide synthesis using phosphoramidite chemistry.
[0769] The synthesis of the above sequences are performed at lum scale in 96 well plates. The amidite solutions are prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) is used as activator.
[0770] The synthesized sequences are cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. The crude sequences are precipitated using acetone: ethanol (80:20) mix and the pellet re-suspended in 0.02M sodium acetate buffer. Samples from each sequence are analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.
Purification and desalting:
[0771] IncR A siRNA sequences are purified on AKTA explorer purification system using Source 15Q column. A column temperature of 65C is maintained during purification. Sample injection and collection is performed in 96 well (1.8mL -deep well) plates. A single peak corresponding to the full length sequence is collected in the eluent. The purified sequences are desalted on a Sephadex G25 column using AKTA purifier. The desalted sequences are analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The single strands are then submitted for annealing.
Example 4; In vitro screening:
Cell culture and transfections;
[0772] Cell culture and transfection conditions are well known in the art and are chosen according to the necessary experimental conditions for study. In one non limiting example, RKO or Hep3B (ATCC, Manassas, VA) cells are grown to near confluence at 37°C in an atmosphere of 5% C02 in McCoy's or EMEM (respectively) (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transfection is carried out by adding 5μ1 of Opti-MEM to 5μ1 of siRNA duplexes per well into a 96-well plate along with ΙΟμΙ of Opti-MEM plus 0.2μ1 of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad CA. cat # 13778-150) and incubated at room temperature for 15 minutes. 80μ1 of complete growth media without antibiotic containing 2.0 xlO4 Hela cells is then added. Cells are incubated for 24 hours prior to RNA purification. Experiments are performed at 0.1 or ΙΟηΜ final duplex concentration for single dose screens with each of the IncRNA siRNA duplexes. The subset duplexes that show robust silencing in the preliminary screens are assayed over a range of concentrations using serial dilutions to determine their IC50.
Total RNA isolation using MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Forer City CA, part #: AMI 830): [0773] Cells are harvested and lysed in 140μ1 of Lysis/Binding Solution then mixed for 1 minute at 850rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads and Lysis/Binding Enhancer mixture are added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads are washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads are capture again and supernatant removed. Beads are then washed with 150μ1 Wash Solution 2 (Ethanol added), captured and supernatant removed. 50ul of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) is then added to the beads and they are mixed for 10 to 15 minutes. After mixing, ΙΟΟμΙ of RNA Rebinding Solution is added and mixed for 3 minutes. Supernatant is removed and magnetic beads are washed again with 150μ1 Wash Solution 2 and mixed for 1 minute and supernatant is removed completely. The magnetic beads are mixed for 2 minutes to dry before RNA was eluted with 50μ1 of water.
cDNA synthesis using ABI High capacity cDNA reverse transcription kit (Applied Biosvstems. Foster City. CA. Cat #4368813):
[0774] A master mix of 2μ1 10X Buffer, 0.8μ1 25X dNTPs, 2μ1 Random primers, Ιμΐ Reverse Transcriptase, Ιμΐ RNase inhibitor and 3.2μ1 of H20 per reaction are added into ΙΟμΙ total RNA. cDNA is generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) through the following steps: 25°C 10 min, 37°C 120 min, 85°C 5 sec, 4°C hold.
Real time PCR:
[0775] 2μ1 of cDNA are added to a master mix containing 0.5μ1 GAPDH TaqMan Probe (Applied Biosystems Cat # 4326317E), 0.5μ1 CD274 (PD-L1) TaqMan probe (Applied
Biosystems cat # HsOl 12530 l_ml) and 5μ1 Roche Probes Master Mix (Roche Cat #
04887301001) in a total of ΙΟμΙ per well in a LightCycler 480 384 well plate (Roche cat # 0472974001). Real time PCR is done in a LightCycler 480 Real Time PCR machine (Roche). Each duplex is tested in at least two independent transfections. Each transfection is assayed by qPCR in duplicate.
[0776] Real time data are analyzed using the AACt method. Each sample is normalized to GAPDH expression and knockdown assessed relative to cells transfected with a non-targeting duplex. IC50s are defined using a 4 parameter fit model in XLfit.
EXAMPLE 5; Synthesis of cationic lipids.
[0777] Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention may be prepared by known organic synthesis techniques. All substituents are as defined below unless indicated otherwise. [0778] "Alkyl" means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
[0779] "Alkenyl" means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2- butenyl, and the like.
[0780] "Alkynyl" means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3 -methyl- 1 butynyl, and the like.
[0781] "Acyl" means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, -C(=0)alkyl, - C(=0)alkenyl, and -C(=0)alkynyl are acyl groups.
[0782] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom.
Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
[0783] The terms "optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted acyl", and "optionally substituted heterocycle" means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (=0) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, -CN, -ORx, -NRxRy, -NRxC(=0)Ry , -NRxS02Ry, -C(=0)Rx, -C(=0)ORx, -C(=0)NRxRy, -SOnRx and -SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -ORx, heterocycle, -NRxRy, -NRxC(=0)Ry -NRxS02Ry, -C(=0)Rx, -C(=0)ORx,
-C(=0)NRxRy, -SO„Rx and -SO„NRxRy.
[0784] "Halogen" means fluoro, chloro, bromo and iodo.
[0785] In some embodiments, the methods of the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T.W. et ah, Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an "alcohol protecting group" is used. An "alcohol protecting group" is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.
Synthesis of Formula A
[0786] In one embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:
Figure imgf000282_0001
where Rl and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2- Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise. [0787] Scheme 1
Figure imgf000283_0001
[0788] Lipid A, where Ri and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
[0789] Scheme 2
Figure imgf000283_0002
[0790] Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
Synthesis of MC3
[0791] Preparation of DLin-M-C3 -DMA (i.e., (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate) is as follows. A solution of (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61g) and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) is stirred at room temperature overnight. The solution is washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions are dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue is passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product are combined and the solvent removed, yielding a colorless oil (0.54 g).
Synthesis of ALNY-100
[0792] Synthesis of ketal 519 [AL Y- 100] is performed using the following scheme 3 :
Figure imgf000284_0001
[0793] Synthesis of 515:
[0794] To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1L), is added a solution of 514 (lOg, 0.04926mol) in 70 mL of THF slowly at 0 0C under nitrogen atmosphere. After complete addition, reaction mixture is warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction is monitored by TLC. After completion of reaction (by TLC) the mixture is cooled to 0 0C and quenched with careful addition of saturated Na2S04 solution. Reaction mixture is stirred for 4 h at room temperature and filtered off. Residue is washed well with THF. The filtrate and washings are mixed and diluted with 400 mL dioxane and 26 mL cone. HC1 and stirred for 20 minutes at room temperature. The volatilities are stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g IH-NMR (DMSO, 400MHz): δ= 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
Synthesis of 516:
[0795] To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, is added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0 0C under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture is allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture is washed successively with IN HCl solution (1 x 100 mL) and saturated NaHC03 solution (1 x 50 mL). The organic layer is then dried over anhyd. Na2S04 and the solvent is evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 1 lg (89%). IH-NMR (CDC13, 400MHz): δ = 7.36-7.27(m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60(m, 2H), 2.30- 2.25(m, 2H). LC-MS [M+H] -232.3 (96.94%).
[0796] Synthesis of 517A and 517B:
[0797] The cyclopentene 516 (5 g, 0.02164 mol) is dissolved in a solution of 220 mL acetone and water (10: 1) in a single neck 500 mL RBF and to it is added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of Os04 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (~ 3 h), the mixture is quenched with addition of solid Na2S03 and resulting mixture is stirred for 1.5 h at room temperature. Reaction mixture is diluted with DCM (300 mL) and washed with water (2 x 100 mL) followed by saturated NaHC03 (1 x 50 mL) solution, water (1 x 30 mL) and finally with brine (lx 50 mL). Organic phase is dried over an.Na2S04 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which are separated by prep HPLC. Yield: - 6 g crude
[0798] 517A - Peak-1 (white solid), 5.13 g (96%). IH-NMR (DMSO, 400MHz): δ= 7.39- 7.3 l(m, 5H), 5.04(s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47(d, 2H), 3.94-3.93(m, 2H), 2.71(s, 3H), 1.72- 1.67(m, 4H). LC-MS - [M+H]-266.3, [M+NH4 +]-283.5 present, HPLC-97.86%.
Stereochemistry confirmed by X-ray.
[0799] Synthesis of 518:
[0800] Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) is obtained as a colorless oil. IH-NMR (CDC13, 400MHz): δ= 7.35- 7.33(m, 4H), 7.30-7.27(m, 1H), 5.37-5.27(m, 8H), 5.12(s, 2H), 4.75(m, lH), 4.58-4.57(m,2H), 2.78-2.74(m,7H), 2.06-2.00(m,8H), 1.96-1.91(m, 2H), 1.62(m, 4H), 1.48(m, 2H), 1.37-1.25(br m, 36H), 0.87(m, 6H). HPLC-98.65%.
[0801] General Procedure for the Synthesis of Compound 519: [0802] A solution of compound 518 (1 eq) in hexane (15 mL) is added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture is heated at 40°C over 0.5 h then cooled again on an ice bath. The mixture is carefully hydrolyzed with saturated aqueous Na2S04 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR = 130.2, 130.1 (x2), 127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M + H)+ Calc. 654.6, Found 654.6.
[0803] Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA, e.g., siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA -binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the "free" dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 1 10 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 1 10 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
LENGTHY TABLE
[0804] The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site. An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).
[0805] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. SHEET INTENTIONALLY LEFT BLANK

Claims

CLAIMS What is claimed is:
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a IncRNA gene, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a IncRNA gene listed in Table 2, which antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense sequences listed in Table 1.
2. The dsRNA of claim 1, wherein said dsRNA comprises at least one modified nucleotide.
3. The dsRNA of claim 2, wherein at least one of said modified nucleotides is chosen from the group consisting of: a 2'-0-methyl modified nucleotide, a nucleotide comprising a 5'-phosphate group, a locked nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy- modified nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
4. The dsRNA of claim 1, wherein the region of complementarity is at least 17 nucleotides in length.
5. The dsRNA of claim 1, wherein the region of complementarity is between 19 and 21 nucleotides in length.
6. The dsRNA of claim 5, wherein the region of complementarity is 19 nucleotides in length.
7. The dsRNA of claim 1, wherein each strand is no more than 30 nucleotides in length.
8. The dsRNA of claim 1, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.
9. The dsRNA of claim 1, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.
10. The dsRNA of claim 1, further comprising a ligand.
11. The dsRNA of claim 10, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA.
12. The dsRNA of claim 1, wherein the region of complementarity consists of one of the antisense sequences selected from SEQ ID NOs 1-745860.
13. The dsRNA of claim 1, wherein the dsRNA comprises a sense strand consisting of a sense strand sequence selected from Table 1, and an antisense strand consisting of an antisense sequence selected from Table 1.
14. A cell containing the dsRNA of claim 1.
15. A pharmaceutical composition for inhibiting expression of a IncRNA comprising the dsRNA of claim 1.
16. The pharmaceutical composition of claim 1 , further comprising a lipid formulation.
17. The pharmaceutical composition of claim 16, wherein the lipid formulation is a SNALP, or XTC-containing formulation.
18. A method of inhibiting IncRNA expression in a cell, the method comprising:
(a) introducing into the cell the dsRNA of claim 1 ; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the RNA transcript of a IncRNA gene, thereby inhibiting expression of the IncRNA in the cell.
19. The method of claim 18, wherein the IncRNA expression is inhibited by at least 30%.
20. A method of treating a disorder mediated by IncRNA expression comprising
administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of claim 1.
21. The method of claim 20, wherein the subject has a disease, disorder or condition associated with a IncRNA.
22. The method of claim 20, wherein the subject has an infectious disease.
23. The method of claim 22, wherein the infectious disease is a viral, bacterial, fungal, or parasitic disease.
24. The method of claim 23, wherein the viral, bacterial, fungal, or parasitic disease is a chronic infectious disease.
25. The method of claim 21, wherein the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg body weight of the subject.
26. A vector encoding at least one strand of a dsRNA, wherein said dsRNA comprises a region of complementarity to at least a part of an RNA encoding a IncRNA, wherein said dsRNA is 30 base pairs or less in length, and wherein said dsRNA targets a said IncRNA for cleavage.
27. The vector of claim 26, wherein the region of complementarity is at least 15 nucleotides in length.
28. The vector of claim 26, wherein the region of complementarity is 19 to 21 nucleotides in length.
29. A cell comprising the vector of claim 26.
30. A method of altering the path of lineage of a cell comprising,
(a) identifying the cell type of a first cell by determining the RNA population signature of said cell,
(b) contacting said first cell with a IncRNA transcript or modified lncR A transcript and allowing said first cell to undergo cellular division to produce daughter cells,
(c) determining the RNA population signature of the daughter cells produced in (b),
(d) comparing the RNA population signature of the first cell with the daughter cells wherein a difference in the RNA population signatures between the first cell and the daughter cells indicates an alteration in the path of lineage of said first cell.
31. The method of claim 30, further comprising confirming the lineal alteration of said first cells by determining the cell type of the daughter cells.
32. The method of claim 31 , wherein the cell type is determined by measuring cell type specific markers.
33. The method of claim 30, wherein the first cell is a cell of a gamete.
34. The method of claim 30, wherein the first cell is a somatic cell.
35. The method of claim 30, wherein the first cell is a stem cell.
36. The method of claim 35, wherein the stem cell is selected from the group consisting of adult, embryonic, pluripotent, and induced pluripotent.
37. The method of claim 34, wherein the somatic cell is a cell selected from the group consisting of endodermal-derived, ectodermal-derived, and mesodermal-derived.
38. The method of claim 30, wherein the modified IncRNA transcript comprises an RNA molecule having one or more domains, motifs, regions or portions from at least two different wild type or parent IncRNA transcripts.
39. The method of claim 30, wherein the IncRNA transcript or modified IncRNA transcript is encoded in a vector.
40. A synthetic isolated IncRNA transcript variant.
41. The IncRNA transcript variant of claim 40, having one or more structural features which differ from the comparable structural feature in the wild type or parent IncRNA transcript.
42. The IncRNA transcript variant of claim 41, wherein said structural features are selected from the group consisting of one or more surface manifestations, local conformational shapes, folds, loops, half-loops, domains, half-domains, sites, molecular interaction sites and termini.
43. The IncRNA transcript variant of claim 42, wherein a structural feature of a wild type or parent IncRNA transcript is removed.
44. The IncRNA transcript variant of claim 42, wherein a structural feature of a wild type or parent IncRNA transcript is duplicated.
45. The IncRNA transcript variant of claim 42, wherein a structural feature of a wild type or parent IncRNA transcript is swapped with a second structural feature of a IncRNA transcript.
46. The IncRNA transcript variant of claim 45, wherein the second feature is from the same or a different IncRNA transcript.
47. The IncRNA transcript variant of any of claims 42-45, wherein said structural feature is a fold.
48. The IncRNA transcript variant of claim 47, wherein the fold is selected from the group consisting of hairpins, loops and bulges.
49. A method of altering the level of a IncRNA transcript in a cell comprising contacting said cell with a IncRNA Directed Agent (LDA).
50. The method of claim 49, wherein the LDA is selected from the group consisting of iRNA agents, antisense molecules, ribozymes, aptamers, small molecules, antibodies, peptides, proteins, enzymes or fragments thereof, and vitamins.
51. A method of altering the expression of a gene comprising contacting a cell with a synthetic isolated IncRNA transcript or IncRNA transcript variant which interacts with chromatin modifying proteins or protein complexes.
52. A method of altering the epigenetic signature of a cell comprising contacting the cell with an artificial nucleosome protein.
53. A method of altering the methylation status or pattern of a chromosome locus comprising contacting a cell containing said chromosome locus with an LDA or a synthetic IncRNA transcript or a compound comprising a IncRNA transcript structural feature.
54. A method of altering the methylation status or pattern of a nucleosome protein comprising contacting a cell containing said nucleosome protein with an LDA or a synthetic IncRNA transcript or a compound comprising a IncRNA transcript structural feature.
55. The method of claim 54, wherein nucleosome protein is a histone.
56. A method of altering cellular protein trafficking comprising contacting the cell with the IncRNA transcript variant of any of claims 40-48.
57. A synthetic isolated RNA molecule comprising a structural feature of a IncRNA transcript, wherein the IncRNA transcript is selected from the group consisting of IncRNA transcripts of Table 2.
58. The synthetic isolated RNA molecule of claim 57, wherein said structural feature is at least 200 nucleotides in length.
59. The synthetic isolated RNA molecule of claim 58, wherein said structural feature is from about 200 to about 500 nucleotides in length.
60. The synthetic isolated RNA molecule of claim 59, wherein said structural feature is from about 200 to about 300 nucleotides in length.
61. The synthetic isolated RNA molecule of claim 57, wherein said structural feature is from about 50 to about 100 nucleotides in length.
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