EP1750775A2 - Compositions et procedes pour l'amelioration de l'administration d'acides nucleiques dans des cellules et pour la modification de l'expression de genes cibles dans des cellules - Google Patents

Compositions et procedes pour l'amelioration de l'administration d'acides nucleiques dans des cellules et pour la modification de l'expression de genes cibles dans des cellules

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Publication number
EP1750775A2
EP1750775A2 EP05804803A EP05804803A EP1750775A2 EP 1750775 A2 EP1750775 A2 EP 1750775A2 EP 05804803 A EP05804803 A EP 05804803A EP 05804803 A EP05804803 A EP 05804803A EP 1750775 A2 EP1750775 A2 EP 1750775A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
polynucleotide delivery
enhancing polypeptide
histone
sirna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05804803A
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German (de)
English (en)
Inventor
Lishan Chen
Kunyuan Cui
Yuching Chen
Sasha J. Mayer
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Marina Biotech Inc
Original Assignee
MDRNA Inc
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Filing date
Publication date
Application filed by MDRNA Inc filed Critical MDRNA Inc
Publication of EP1750775A2 publication Critical patent/EP1750775A2/fr
Withdrawn legal-status Critical Current

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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
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the invention relates to methods and compositions for delivering nucleic acids into cells. More specifically, the invention relates to procedures and preparations for delivering double-stranded polynucleotides into cells to modify expression of target genes to alter a phenotype, such as a disease state or potential, of the cells.
  • RNAi RNA interference
  • Cells can be transfected to uptake a functional nucleic acid from an exogenous source using a variety of techniques and materials.
  • the most commonly used transfection methods are calcium phosphate transfection, and electroporation.
  • a variety of other methods for tranducing cells to deliver exogenous DNA or RNA molecules have been developed, including viral-mediated transduction, cationic lipid or liposomal delivery, and numerous methods that target mechanical or biochemical membrane disruption/penetration (e.g., using detergents, microinjection, or particle guns).
  • RNA interference is a process of sequence-specific post transcriptional gene silencing in cells initiated by a double-stranded (ds) polynucleotide, usually a dsRNA, that is homologous in sequence to a portion of a targeted messenger RNA (mRNA).
  • ds double-stranded polynucleotide
  • mRNA messenger RNA
  • a suitable dsRNA into cells leads to destruction of endogenous, cognate mRNAs (i.e., mRNAs that share substantial sequence identity with the introduced dsRNA).
  • the dsRNA molecules are cleaved by an RNase III family nuclease called dicer into short-interfering RNAs (siRNAs), which are 19-23 nucleotides (nt) in length.
  • RNA-induced silencing complex a multicomponent nuclease complex known as the RNA-induced silencing complex or "RISC".
  • the RISC identifies mRNA substrates through their homology to the siRNA, and effectuates silencing of gene expression by binding to and destroying the targeted mRNA.
  • RNA interference is emerging a promising technology for modifying expression of specific genes in plant and animal cells, and is therefore expected to provide useful tools to treat a wide range of diseases and disorders amenable to treatment by modification of endogenous gene expression.
  • siRNAs and other small inhibitory nucleic acids are limited by poor efficiency and or high toxicity of the delivery reagents.
  • Figure 1 illustrates peptide-mediated uptake of siNAs complexed or conjugated to a polynucleotide delivery-enhancing polypeptide of the invention.
  • Figure 2 further illustrates peptide-mediated uptake of siNAs complexed or conjugated to a polynucleotide delivery-enhancing polypeptide of the invention.
  • Figure 3 shows paw data for siRNA/peptide injected mice which demonstrate delayed RA progression in the treated mice comparable to that exhibited by Ramicade- treated subjects.
  • Figure 4 provides results of uptake efficacy and viability studies in mouse fibroblasts for PN73 rationally-designed derivative polynucleotide delivery-enhancing polypeptides of the invention.
  • the present invention satisfies these needs and fulfills additional objects and advantages by providing novel compositions and methods that employ a short interfering nucleic acid (siNA), or a precursor thereof, in combination with a polynucleotide delivery-enhancing polypeptide.
  • the polynucleotide delivery-enhancing polypeptide is a natural or artificial polypeptide selected for its ability to enhance intracellular delivery or uptake of polynucleotides, including siNAs and their precursors.
  • the siNA may be admixed or complexed with, or conjugated to, the polynucleotide delivery-enhancing polypeptide to form a composition that enhances intracellular delivery of the siNA as compared to delivery resulting from contacting the target cells with a naked siNA (i.e., siNA without the delivery-enhancing polypeptide present).
  • the polynucleotide delivery-enhancing polypeptide is a histone protein, or a polypeptide or peptide fragment, derivative, analog, or conjugate thereof.
  • the siNA is admixed, complexed or conjugated with one or more full length histone proteins or polypeptides corresponding at least in part to a partial sequence of a histone protein, for example of one or more of the following histones: histone HI, histone H2A, histone H2B, histone H3 or histone H4, or one or more polypeptide fragments or derivatives thereof comprising at least a partial sequence of a histone protein, typically at least 5-10 or 10-20 contiguous residues of a native histone protein.
  • the siRNA/histone mixture, complex or conjugate is substantially free of amphipathic compounds.
  • the siNA that is admixed, complexed, or conjugated with the histone protein or polypeptide will comprise a double-stranded double-stranded RNA, for example a double-stranded RNA that has 30 or fewer nucleotides, and is a short interfering RNA (siRNA).
  • the histone polynucleotide delivery-enhancing polypeptide comprises a fragment of histone H2B, as exemplified by the polynucleotide delivery-enhancing polypeptide designated PN73 described herein below.
  • the polynucleotide delivery-enhancing polypeptide may be pegylated to improve stability and/or efficacy, particularly in the context of in vivo administration.
  • the polynucleotide delivery- enhancing polypeptide is selected or rationally designed to comprise an amphipathic amino acid sequence.
  • useful polynucleotide delivery-enhancing polypeptides may be selected which comprise a plurality of non-polar or hydrophobic amino acid residues that form a hydrophobic sequence domain or motif, linked to a plurality of charged amino acid residues that form a charged sequence domain or motif, yielding an amphipathic peptide.
  • the polynucleotide delivery-enhancing polypeptide is selected to comprise a protein transduction domain or motif, and a fusogenic peptide domain or motif.
  • a protein transduction domain is a peptide sequence that is able to insert into and preferably transit through the membrane of cells.
  • a fusogenic peptide is a peptide that is able destabilize a lipid membrane, for example a plasma membrane or membrane surrounding an endosome, which may be enhanced at low pH.
  • Exemplary fusogenic domains or motifs are found in a broad diversity of viral fusion proteins and in other proteins, for example fibroblast growth factor 4 (FGF4).
  • FGF4 fibroblast growth factor 4
  • a protein transduction domain is employed as a motif that will facilitate entry of the nucleic acid into a cell through the plasma membrane.
  • the transported nucleic acid will be encapsulated in an endosome.
  • the interior of endosomes has a low pH resulting in the fusogenic peptide motif destabilizing the membrane of the endosome.
  • the destabilization and breakdown of the endosome membrane allows for the release of the siNA into the cytoplasm where the siNA can associate with a RISC complex and be directed to its target mRNA.
  • protein transduction domains for optional incorporation into polynucleotide delivery-enhancing polypeptides of the invention include:
  • TAT protein transduction domain (SEQ ID NO: 1 ) KRRQRRR; 2.
  • Penetratin PTD (SEQ ID NO: 2) RQIKIWFQNRRMKWKK;
  • Kaposi FGF signal sequences (SEQ ID NO: 4) AAVALLPAVLLALLAP, and SEQ ID NO: 5) AAVLLPNLLPVLLAAP;
  • Caiman crocodylus Ig(v) light chain (SEQ ID NO: 8) MGLGLHLLVLAAALQGA;
  • Transportan (SEQ ID NO: 10) GWTLNSAGYLLKINLKALAALAKKIL;
  • Loligomer (SEQ ID NO: 11) TPPKKKRKVEDPKKKK; 11. Arginine peptide (SEQ ID NO: 12) RRRRRRR; and 12. Amphiphilic model peptide (SEQ ID NO: 13) KLALKLALKALKAALKLA.
  • viral fusion peptides fusogenic domains for optional incorporation into polynucleotide delivery-enhancing polypeptides of the invention include:
  • Influenza HA2 (SEQ ID NO: 14) GLFGAIAGFIENGWEG;
  • Sendai Fl (SEQ ID NO: 15) FFGAVIGTIALGVATA; 3. Respiratory Syncytial virus Fl (SEQ ID NO: 16) FLGFLLGVGSAIASGV;
  • HIV gp41 (SEQ ID NO: 17) GVFVLGFLGFLATAGS;
  • Ebola GP2 (SEQ ID NO: 18) GAAIGLAWIPYFGPAA.
  • polynucleotide delivery- enhancing polypeptides are provided that incorporate a DNA-binding domain or motif which facilitates polypeptide-siNA complex formation and/or enhances delivery of siNAs within the methods and compositions of the invention.
  • Exemplary DNA binding domains in this context include various "zinc finger" domains as described for DNA- binding regulatory proteins and other proteins identified in Table 1, below (see, e.g., Simpson et al, J. Biol. Chem. 278:28011-28018, 2003).
  • Table 1 Exemplary zinc finger motifs of different DNA-binding proteins
  • polynucleotide delivery-enhancing polypeptides may be rationally designed and constructed by combining any of the foregoing structural elements, domains or motifs into a single polypeptide effective to mediate enhanced delivery of siNAs into target cells.
  • a protein transduction domain of the TAT polypeptide was fused to the N-terminal 20 amino acids of the influenza virus hemagglutinin protein, termed HA2, to yield one exemplary polynucleotide delivery-enhancing polypeptide herein.
  • polynucleotide delivery-enhancing polypeptide constructs are provided in the instant disclosure, evincing that the concepts of the invention are broadly applicable to create and use a diverse assemblage of effective polynucleotide delivery-enhancing polypeptides for enhancing siNA delivery.
  • Yet additional exemplary polynucleotide delivery-enhancing polypeptides within the invention may be selected from the following peptides: WETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27); GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO: 29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), Poly Lys-Trp, 4:1, MW 20,000- 50,000; and Poly Orn-Trp, 4:1, MW 20,000-50,000.
  • Additional polynucleotide delivery- enhancing polypeptides that are usefiil within the compositions and methods herein comprise all or part of the mellitin protein sequence. Still other exemplary polynucleotide delivery-enhancing polypeptides are identified in the examples below. Any one or combination of these peptides may be selected or combined to yield effective polynucleotide delivery-enhancing polypeptide reagents to induce or facilitate intracellular delivery of siNAs within the methods and compositions of the invention.
  • the mixture, complex or conjugate comprising a siRNA and a polynucleotide delivery-enhancing polypeptide can be optionally combined with (e.g., admixed or complexed with) a cationic lipid, such as LIPOFECTIN®.
  • a cationic lipid such as LIPOFECTIN®
  • siRNA/polynucleotide delivery- enhancing polypeptide complex or conjugate will exhibit even greater activity for mediating siNA delivery and gene silencing when admixed or complexed with a cationic lipid, such as lipofectin.
  • a cationic lipid such as lipofectin.
  • the siRNA and peptide may be mixed together first in a suitable medium such as a cell culture medium, after which the cationic lipid is added to the mixture to form a siRNA/delivery peptide/cationic lipid composition.
  • the peptide and cationic lipid can be mixed together first in a suitable medium such as a cell culture medium, whereafter the siRNA can be added to form the siRNA delivery peptide/cationic lipid composition.
  • a suitable medium such as a cell culture medium
  • useful cationic lipids within these aspects of the invention include N- [l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, l,2-bis(oleoyloxy)-3- 3-(trimethylammonium)propane, l,2-dimyristyloxypropyl-3- dimethylhydroxyethylammonium bromide, and dimethyldioctadecylammomum bromide, 2,3-dioleyloxy-N-[2(sperrmnecarboxamido)emyl]-N,N-dimethyl-l-propanaminium trifluoracetate, l,3-
  • DOTMA N-[l-(2,3-dioleoyloxy)propyl]- N,N,N-trimethyl ammonium chloride
  • DOTAP l,2-bis(oleoyloxy)-3,3- (trimethylammonium)propane
  • DMRIE 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide
  • DDAB dimethyl dioctadecyl ammonium bromide
  • Polyvalent cationic lipids include lipospermines, specifically DOSPA (2,3-dioleyloxy-N- [2(spe ⁇ mnecarboxamido)ethyl]-N,N-dimethyl-l -propanamini um trifluoro-acetate) and DOSPER (l,3-dioleoyloxy-2-(6carboxy spermyl)-propyl-amid, and the di- and tetra- alkyl-tetra-methyl spermines, including but not limited to TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethlytetralauryl spermine), TMTMS (tetramethyltetramyristyl spermine) and TMDOS (tetramethyldioleyl spermine) DOGS (dioctadecyl-amidog
  • Cationic lipids are optionally combined with non-cationic lipids, particularly neutral lipids, for example lipids such as DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol.
  • DOPE dioleoylphosphatidylethanolamine
  • DPhPE diphytanoylphosphatidylethanolamine
  • a cationic lipid composition composed of a 3:1 (w/w) mixture of DOSPA and DOPE or a 1:1 (w/w) mixture of DOTMA and DOPE (LIPOFECTIN®, Invitrogen) are generally useful in transfecting compositions of this invention.
  • Preferred transfection compositions are those which induce substantial transfection of a higher eukaryotic cell line.
  • the instant invention features compositions comprising a small nucleic acid molecule, such as short interfering nucleic acid (siNA), a short interfering RNA (siRNA), a double-stranded RNA (dsRNA), micro-RNA (mRNA), or a short hairpin RNA (shRNA), admixed or complexed with, or conjugated to, a polynucleotide delivery-enhancing polypeptide.
  • siNA short interfering nucleic acid
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • mRNA micro-RNA
  • shRNA short hairpin RNA
  • short interfering nucleic acid refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference "RNAi” or gene silencing in a sequence-specific manner.
  • the siNA is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule for downregulating expression, or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to (i.e., which is substantially identical in sequence to) the target nucleic acid sequence or portion thereof.
  • siNA means a small interfering nucleic acid, for example a siRNA, that is a short-length double-stranded nucleic acid (or optionally a longer precursor thereof), and which is not unacceptably toxic in target cells.
  • siNAs within the invention will in certain embodiments be optimized at a length of approximately 21 to 23 bp long.
  • length of useful siNAs including siRNAs.
  • siNAs can initially be presented to cells in a precursor form that is substantially different than a final or processed form of the siNA that will exist and exert gene silencing activity upon delivery, or after delivery, to the target cell.
  • Precursor forms of siNAs may, for example, include precursor sequence elements that are processed, degraded, altered, or cleaved at or following the time of delivery to yield a siNA that is active within the cell to mediate gene silencing.
  • useful siNAs within the invention will have a precursor length, for example, of approximately 100-200 base pairs, 50-100 base pairs, or less than about 50 base pairs, which will yield an active, processed siNA within the target cell.
  • a useful siNA or siNA precursor will be approximately 10 to 49 bp, 15 to 35 bp, or about 21 to 30 bp in length.
  • polynucleotide delivery- enhancing polypeptides are used to facilitate delivery of larger nucleic acid molecules than conventional siNAs, including large nucleic acid precursors of siNAs.
  • the methods and compositions herein may be employed for enhancing delivery of larger nucleic acids that represent "precursors" to desired siNAs, wherein the precursor amino acids may be cleaved or otherwise processed before, during or after delivery to a target cell to form an active siNA for modulating gene expression within the target cell.
  • a siNA precursor polynucleotide may be selected as a circular, single-stranded polynucleotide, having two or more loop structures and a stem comprising self- complementary sense and antisense regions, wherem the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi.
  • dsRNAs longer than 30 base pairs can activate the dsRNA-dependent kinase PKR and 2'-5'-oligoadenylate synthetase, normally induced by interferon.
  • the activated PKR inhibits general translation by phosphorylation of the translation factor eukaryotic initiation factor 2 ⁇ (eIF2 ⁇ ), while 2'-5'-oligoadenylate synthetase causes nonspecific mRNA degradation via activation of RNase L.
  • eIF2 ⁇ translation factor eukaryotic initiation factor 2 ⁇
  • the siNAs of the present invention avoid activation of the interferon response.
  • siRNA can mediate selective gene silencing in the mammalian system.
  • Hairpin RNAs with a short loop and 19 to 27 base pairs in the stem, also selectively silence expression of genes that are homologous to the sequence in the double-stranded stem.
  • Mammalian cells can convert short hairpin RNA into siRNA to mediate selective gene silencing.
  • RISC mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
  • the siNAs can be delivered as single or multiple transcription products expressed by a polynucleotide vector encoding the single or multiple siNAs and directing their expression within target cells.
  • the double-stranded portion of a final transcription product of the siRNAs to be expressed within the target cell can be, for example, 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bp long.
  • double-stranded portions of siNAs are not limited to completely paired nucleotide segments, and may contain nonpairing portions due to mismatch (the corresponding nucleotides are not complementary), bulge (lacking in the corresponding complementary nucleotide on one strand), overhang, and the like.
  • Nonpairing portions can be contained to the extent that they do not interfere with siNA formation.
  • a "bulge" may comprise 1 to 2 nonpairing nucleotides, and the double-stranded region of siNAs in which two strands pair up may contain from about 1 to 7, or about 1 to 5 bulges.
  • mismatch portions contained in the double-stranded region of siNAs may be present in numbers from about 1 to 7, or about 1 to 5. Most often in the case of mismatches, one of the nucleotides is guanine, and the other is uracil. Such mismatching may be attributable, for example, to a mutation from C to T, G to A, or mixtures thereof, in a corresponding DNA coding for sense RNA, but other cause are also contemplated. Furthermore, in the present invention the double-stranded region of siNAs in which two strands pair up may contain both bulge and mismatched portions in the approximate numerical ranges specified.
  • the terminal structure of siNAs of the invention may be either blunt or cohesive (overhanging) as long as the siNA retains its activity to silence expression of target genes.
  • the cohesive (overhanging) end structure is not limited only to the 3' overhang as reported by others.
  • the 5' overhanging structure may be included as long as it is capable of inducing a gene silencing effect such as by RNAi.
  • the number of overhanging nucleotides is not limited to reported limits of 2 or 3 nucleotides, but can be any number as long as the overhang does not impair gene silencing activity of the siNA.
  • overhangs may comprise from about 1 to 8 nucleotides, more often from about 2 to 4 nucleotides.
  • the total length of siNAs having cohesive end structure is expressed as the sum of the length of the paired double-stranded portion and that of a pair comprising overhanging single-strands at both ends. For example, in the exemplary case of a 19 bp double-stranded RNA with 4 nucleotide overhangs at both ends, the total length is expressed as 23 bp. Furthermore, since the overhanging sequence may have low specificity to a target gene, it is not necessarily complementary (antisense) or identical (sense) to the target gene sequence.
  • the siNA may contain low molecular weight structure (for example a natural RNA molecule such as tRNA, rRNA or viral RNA, or an artificial RNA molecule), for example, in the overhanging portion at one end.
  • the terminal structure of the siNAs may have a stem-loop structure in which ends of one side of the double-stranded nucleic acid are connected by a linker nucleic acid, e.g., a linker RNA.
  • the length of the double-stranded region (stem-loop portion) can be, for example, 15 to 49 bp, often 15 to 35 bp, and more commonly about 21 to 30 bp long.
  • the length of the double-stranded region that is a final transcription product of siNAs to be expressed in a target cell may be, for example, approximately 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bp long.
  • linker segments there is no particular limitation in the length of the linker as long as it does not hinder pairing of the stem portion.
  • the linker portion may have a clover-leaf tRNA structure.
  • RNA with no loop structure may have a low molecular weight RNA.
  • these low molecular weight RNAs may include a natural RNA molecule, such as tRNA, rRNA or viral RNA, or an artificial RNA molecule.
  • the siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5'-phosphate (see for example Martinez et al.Cell, 110: 563-574 (2002) and Schwarz et al, Molecular Cell, 10: 537-568(2002), or 5',3'-diphosphate.
  • a terminal phosphate group such as a 5'-phosphate
  • siNA molecule is not limited to molecules containing only naturally-occurring RNA or DNA, but also encompasses chemically-modified nucleotides and non-nucleotides.
  • the short interfering nucleic acid molecules of the invention lack 2'-hydroxy (2'-OH) containing nucleotides.
  • short interfering nucleic acids do not require the presence of nucleotides having a 2'-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group).
  • siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
  • siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
  • siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro- RNA (mRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others.
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • mRNA micro- RNA
  • shRNA short hairpin RNA
  • siRNA short interfering oligonucleotide
  • short interfering nucleic acid short interfering modified oligonucleotide
  • ptgsRNA post-transcriptional gene silencing RNA
  • siNA molecules for use within the invention may comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic intercations, and/or stacking interactions.
  • Antisense RNA is an RNA strand having a sequence complementary to a target gene mRNA, and thought to induce RNAi by binding to the target gene mRNA.
  • Sense RNA has a sequence complementary to the antisense RNA, and annealed to its complementary antisense RNA to form siRNA.
  • RNAi constructs are generic term used throughout the specification to include small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleaved in vivo to form siRNAs.
  • RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving rise to transcripts which form dsRNAs or hairpin RNAs in cells, and/or transcripts which can produce siRNAs in vivo.
  • the siRNA include single strands or double strands of siRNA.
  • siHybrid molecule is a double-stranded nucleic acid that has a similar function to siRNA.
  • an siHybrid is comprised of an RNA strand and a DNA strand.
  • the RNA strand is the antisense strand as that is the strand that binds to the target mRNA.
  • the siHybrid created by the hybridization of the DNA and RNA strands have a hybridized complementary portion and preferably at least one 3 Overhanging end.
  • siNAs for use within the invention can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs).
  • the antisense strand may comprise a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand may comprise a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid-based or non-nucleic acid-based linker(s).
  • siNAs for intracellular delivery according to the methods and compositions of the invention can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • Non-limiting examples of chemical modifications that can be made in an siNA include without limitation phosphorothioate internucleotide linkages, 2'- deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base” nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation.
  • These chemical modifications, when used in various siNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds.
  • the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously.
  • the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum.
  • certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule.
  • the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3'-end of said antisense region.
  • the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5'-end of said antisense region.
  • the 3'-terrninal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically- modified at a nucleic acid sugar, base, or backbone.
  • the 3'-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides.
  • the 3'-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.
  • the invention features a chemically- modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand.
  • the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands.
  • the phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands.
  • the siNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends of the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.
  • An siNA molecule may be comprised of a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.
  • a circular siNA molecule contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable.
  • a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3 '-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising about 2 nucleotides.
  • Modified nucleotides present in siNA molecules, preferably in the antisense strand of the siNA molecules, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides.
  • the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer- Verlag ed., 1984).
  • a Northern conformation e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer- Verlag ed., 1984.
  • chemically modified nucleotides present in the siNA molecules of the invention preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.
  • Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene- (D-ribofuranosyl) nucleotides); 2'-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio- ethyl, 2'-deoxy-2'-fluoro micleotides. 2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides, and 2'-O-methyl nucleotides.
  • LNA locked nucleic acid
  • MOE 2'-methoxyethoxy
  • the sense strand of a double stranded siNA molecule may have a terminal cap moiety such as an inverted deoxyabaisc moiety, at the 3'-end, 5'-end, or both 3' and 5'- ends of the sense strand.
  • conjugates include conjugates and ligands described in Vargeese et al., U.S. Application Serial No. 10/427,160, filed April 30, 2003, incorporated by reference herein in its entirety, including the drawings, hr another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker.
  • the conjugate molecule is attached at the 3 '-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3'-end and 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof.
  • a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell.
  • the conjugate molecule attached to the chemically- modified siNA molecule is a poly ethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically- modified siNA molecules are described in Vargeese et al., U.S. Patent Application Publication No. 20030130186, published July 10, 2003, and U.S. Patent Application Publication No. 20040110296, published June 10, 2004.
  • siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity.
  • one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.
  • a siNA further maybe further comprised of a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA.
  • a nucleotide linker can be a linker of >2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.
  • the nucleotide linker can be a nucleic acid aptamer.
  • aptamer or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting.
  • an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid.
  • the target molecule can be any molecule of interest.
  • the aptamer can be used to bind to a ligand- binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. [See, for example, Gold et al, Annu. Rev. Biochem., 64: 763 (1995); Brody and Gold, J. Biotechnol., 74: 5 (2000); Sun, Curr. Opin.
  • a non-nucleotide linker may be comprised of an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units).
  • non-nucleotide further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosme, guanine, cytosine, uracil or thyrnine, for example at the Cl position of the sugar.
  • the synthesis of a siNA molecule of the invention comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule.
  • synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis.
  • synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.
  • Oligonucleotides are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3- 19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No.
  • RNA including certain siNA molecules of the invention, follows general procedures as described, for example, in Usman et al., 1987, j. Am. Chem. Soc, 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59.
  • Nucleic acid molecules and polynucleotide delivery-enhancing polypeptides can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, administration within formulations that comprise the siNA and polynucleotide delivery-enhancing polypeptide alone, or that further comprise one or more additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, and the like.
  • the siNA and/or the polynucleotide delivery-enhancing polypeptide can be encapsulated in liposomes, admimstered by iontophoresis, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (see e.g., O'Hare and Normand, International PCT Publication No. WO 00/53722).
  • a nucleic acid/peptide/vehicle combination can be locally delivered by direct injection or by use of an infusion pump.
  • compositions of the instant invention can be effectively employed as pharmaceutical agents.
  • Pharmaceutical agents prevent, modulate the occurrence or severity of, or treat (alleviate one or more symptom(s) to a detectable or measurable extent) of a disease state or other adverse condition in a patient.
  • the invention provides pharmaceutical compositions and methods featuring the presence or administration of one or more polynucleic acid(s), typically one or more siNAs, combined, complexed, or conjugated with a polynucleotide delivery-enhancing polypeptide, optionally formulated with a pharmaceutically-acceptable carrier, such as a diluent, stabilizer, buffer, and the like.
  • a pharmaceutically-acceptable carrier such as a diluent, stabilizer, buffer, and the like.
  • the siNA will target a gene that is expressed at an elevated level as a causal or contributing factor associated with the subject disease state or adverse condition.
  • the siNA will effectively downregulate expression of the gene to levels that prevent, alleviate, or reduce the severity or recurrence of one or more associated disease symptoms.
  • down regulation of the target gene will nonetheless result in a therapeutic result by lowering gene expression (i.e., to reduce levels of a selected mRNA and/or protein product of the target gene).
  • siNAs of the invention may be targeted to lower expression of one gene, which can result in upregulation of a
  • compositions and methods of the invention are useful as therapeutic tools to regulate expression of tumor necrosis factor- ⁇ (TNF- ⁇ ) to treat or prevent symptoms of rheumatoid arthritis (RA).
  • TNF- ⁇ tumor necrosis factor- ⁇
  • RA rheumatoid arthritis
  • the invention further provides compounds, compositions, and methods useful for modulating expression and activity of TNF- ⁇ by RNA interference (RNAi) using small nucleic acid molecules.
  • RNAi RNA interference
  • the invention provides small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA), and short hairpin RNA (shRNA) molecules, and related methods, that are effective for modulating expression of TNF- ⁇ and/or TNF- ⁇ genes to prevent or alleviate symptoms of RA in mammalian subjects.
  • siNA short interfering nucleic acid
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • mRNA micro-RNA
  • shRNA short hairpin RNA
  • siNA molecules of the instant invention thus provide useful reagents and methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.
  • This siNAs of the present invention may be administered in any form, for example transdermally or by local injection (e.g., local injection at sites of psoriatic plaques to treat psoriasis, or into the joints of patients afflicted with psoriatic arthritis or RA).
  • the invention provides formulations and methods to administer therapeutically effective amounts of siNAs directed against of a mRNA of TNF- ⁇ , which effectively down-regulate the TNF- ⁇ RNA and thereby reduce or prevent one or more TNF- ⁇ -associated inflammatory conditions).
  • Comparable methods and compositions are provided that target expression of one or more different genes associated with a selected disease condition in animal subjects, including any of a large number of genes whose expression is known to be aberrantly increased as a causal or contributing factor associated with the selected disease condition.
  • siNA/polynucleotide delivery-enhancing polypeptide mixtures of the invention can be administered in conjunction with other standard treatments for a targeted disease condition, for example in conjunction with therapeutic agents effective against inflammatory diseases, such as RA or psoriasis.
  • therapeutic agents effective against inflammatory diseases such as RA or psoriasis.
  • combmatorially useful and effective agents in this context include non-steroidal antiinflammatory drugs (NSAIDs), methotrexate, gold compounds, D-penicillamine, the antimalarials, sulfasalazine, glucocorticoids, and other TNF- ⁇ neutralizing agents such as infliximab and entracept.
  • Negatively charged polynucleotides of the invention can be administered to a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.
  • RNA or DNA can be administered to a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.
  • standard protocols for formation of liposomes can be followed.
  • the compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art.
  • the present invention also includes pharmaceutically acceptable formulations of the compositions described herein.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble.
  • systemic administration in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.
  • Administration routes which lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue.
  • the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.
  • a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endofhelial system (RES).
  • RES reticular endofhelial system
  • a liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.
  • pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.
  • agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS [Jolliet-Riant and Tillement, Fundam. Clin. Pharmacol, 13:16-26 (1999)]; biodegradable polymers, such as poly (DL-lactide- coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D F et al, Cell Transplant, 8: 47-58 (1999)] (Alkermes, Inc.
  • nanoparticles such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23: 941-949, (1999)].
  • Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al, J. Pharm. Sci., 87: 1308-1315
  • the present invention also includes compositions prepared for storage or administration, which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid.
  • antioxidants and suspending agents may be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence of, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl- methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
  • dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin,
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations.
  • compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.
  • Pharmaceutical compositions of the invention can also be in the form of oil-in- water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, nalirrally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavoring agents.
  • the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • a non-toxic parentally acceptable diluent or solvent for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono-or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of inj ectables .
  • the siNAs can also be administered in the form of suppositories, e.g., for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials include cocoa butter and polyethylene glycols.
  • the siNAs can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H.
  • SiNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography and resuspended in water. Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency. See e.g., Eckstein et al, International Publication No. WO 92/07065; Perrault et al, Nature 344: 565 (1990); Pieken et al, Science 253, 314 (1991); Usman and Cedergren, Trends in Biochem. Sci. 17: 334 (1992); Usman et al, International Publication No.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O- allyl, 2'-H, nucleotide base modifications.
  • nuclease resistant groups for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O- allyl, 2'-H, nucleotide base modifications.
  • the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • Nucleic acid molecules can be admimstered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al, Bioconjugate Chem., 10: 1068-1074 (1999); Wang et al, International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)ac- id (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent Application Publication No.
  • nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.
  • Direct injection of the nucleic acid molecules of the invention can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al, Clin. Cancer Res., 5: 2330-2337 (1999) and Barry et al, International PCT Publication No. WO 99/31262.
  • the molecules of the instant invention can be used as pharmaceutical agents.
  • ligand refers to any compound or molecule, such as a drug, peptide, hormone, or neuroxransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly.
  • the receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercullular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.
  • asymmetric hairpin as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non- nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop.
  • an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a T-cell (e.g.
  • the asymmetric hairpin siNA molecule can also comprise a 5'-terminal phosphate group that can be chemically modified.
  • the loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.
  • asymmetric duplex as used herein is meant a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex.
  • an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a T-cell (e.g. about 19 to about 22 (e.g.
  • modulate gene expression is meant that the expression of a target gene is upregulated or downregulated, which can include upregulation or downregulation of. mRNA levels present in a cell, or of mRNA translation, or of synthesis of protein or protein subunits, encoded by the target gene.
  • Modulation of gene expression can be determined also be the presence, quantity, or activity of one or more proteins or protein subunits encoded by the target gene that is up regulated or down regulated, such that expression, level, or activity of the subject protein or subunit is greater than or less than that which is observed in the absence of the modulator (e.g., a siRNA).
  • the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
  • inhibit By “inhibit”, “down-regulate”, or “reduce” expression, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or level or activity of one or more proteins or protein subunits encoded by a target gene, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention.
  • inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule.
  • inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches.
  • inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.
  • Gene "silencing” refers to partial or complete loss-of-function through targeted inhibition of gene expression in a cell and may also be referred to as “knock down”. Depending on the circumstances and the biological problem to be addressed, it may be preferable to partially reduce gene expression. Alternatively, it might be desirable to reduce gene expression as much as possible. The extent of silencing may be determined by methods known in the art, some of which are summarized in International Publication No. WO 99/32619.
  • quantitation of gene expression permits detection of various amounts of inhibition that may be desired in certain embodiments of the invention, including prophylactic and therapeutic methods, which will be capable of knocking down target gene expression, in terms of mRNA levels or protein levels or activity, for example, by equal to or greater than 10%, 30%, 50%, 75% 90%, 95% or 99% of baseline (i.e., normal) or other control levels, including elevated expression levels as may be associated with particular disease states or other conditions targeted for therapy.
  • baseline i.e., normal
  • control levels including elevated expression levels as may be associated with particular disease states or other conditions targeted for therapy.
  • inhibitor expression of a target gene refers to the ability of a siNA of the invention to initiate gene silencing of the target gene.
  • samples or assays of the organism of interest or cells in culture expressing a particular construct are compared to control samples lacking expression of the construct.
  • Control samples (lacking construct expression) are assigned a relative value of 100%. Inhibition of expression of a target gene is achieved when the test value relative to the control is about 90%, often 50%, and in certain embodiments 25-0%.
  • Suitable assays include, e.g., examination of protein or mRNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • subject is meant an organism, tissue, or cell, which may include an organism as the subject or as a donor or recipient of explanted cells or the cells that are themselves subjects for siNA delivery.
  • Subject therefore may refers to an organism, organ, tissue, or cell, including in vitro or ex vivo organ, tissue or cellular subjects, to which the nucleic acid molecules of the invention can be administered and enhanced by polynucleotide delivery-enhancing polypeptides described herein.
  • Exemplary subjects include mammalian individuals or cells, for example human patients or cells.
  • cell is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human.
  • the cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
  • the cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing.
  • the cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.
  • vectors is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a .beta.-D-ribo-furanose moiety.
  • RNA double-stranded RNA
  • single-stranded RNA isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinanfly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • highly conserved sequence region is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.
  • siNA region is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule.
  • the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.
  • antisense region is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence.
  • the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule.
  • target nucleic acid is meant any nucleic acid sequence whose expression or activity is to be modulated.
  • the target nucleic acid can be DNA or RNA.
  • nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonuelcotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively).
  • “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • the term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them.
  • Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).
  • acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (Cl, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.
  • biodegradable refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
  • biologically active molecule refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system.
  • Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof.
  • therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A
  • Biologically active molecules of the invention also include molecules capable of modulating the pharmacokineti.es and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
  • lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
  • phospholipid refers to a hydrophobic molecule comprising at least one phosphorus group.
  • a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.
  • cap structure is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell.
  • the cap may be present at the 5'-terminus (5'-cap) or at the 3'-terminal (3'-cap) or may be present on both termini.
  • the 5'-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5- dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety; 3 '-3 '-inverted abasic mo
  • Non-limiting examples of the 3'-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4',5'-methylene nucleotide; l-(beta-D- eiythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3
  • non-nucleotide any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1 '-position.
  • nucleotide as used herein is as recognized in the art to include natural bases
  • Nucleotides generally comprise a base, sugar and a phosphate group.
  • the nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT
  • base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5- alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5- halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6- methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090;
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents.
  • target site is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.
  • detecttable level of cleavage is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA.
  • biological system is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human, animal, plant, insect, bacterial, viral or other sources, wherein the system comprises the components required for RNAi acitivity.
  • biological system includes, for example, a cell, tissue, or organism, or extract thereof.
  • biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.
  • biodegradable linker refers to a nucleic acid or non- nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention.
  • the biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type.
  • the stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'-modified or base modified nucleotides.
  • the biodegradable nucleic acid linker molecule can be a dimer, trirner, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage.
  • the biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
  • abasic sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, see for example Adamic et al., U.S. Pat. No. 5,998,203.
  • unmodified nucleoside is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1' carbon of .beta.-D-ribo-furanose.
  • modified nucleoside is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
  • Non-limiting examples of modified nucleotides are shown by Formulae I- VII and/or other modifications described herein.
  • amino is meant 2'-NH 2 or 2'-O ⁇ NH 2 , which can be modified or unmodified.
  • modified groups are described, for example, in Eckstein et al, U.S. Pat. No. 5,672,695 and Matulic-Adamic et al, U.S. Patent. No. 6,248,878.
  • the siNA molecules can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues.
  • the nucleic acid or nucleic acid complexes can be locally admimstered to through injection, infusion pump or stent, with or without their incorporation in biopolymers.
  • polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention, to the polynucleotide delivery-enhancing polypeptide, or both.
  • the attached PEG can be any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).
  • the sense region can be connected to the antisense region via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker.
  • Inverted repeat refers to a nucleic acid sequence comprising a sense and an antisense element positioned so that they are able to form a double stranded siRNA when the repeat is transcribed.
  • the inverted repeat may optionally include a linker or a heterologous sequence such as a self-cleaving ribozyme between the two elements of the repeat.
  • the elements of the inverted repeat have a length sufficient to form a double stranded RNA.
  • each element of the inverted repeat is about 15 to about 100 nucleotides in length, preferably about 20-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form.
  • nucleic acids containing known nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • “Large double-stranded RNA” refers to any double-stranded RNA having a size greater than about 40 base pairs (bp) for example, larger than 100 bp or more particularly larger than 300 bp.
  • the sequence of a large dsRNA may represent a segment of a mRNA or the entire mRNA. The maximum size of the large dsRNA is not limited herein.
  • the double-stranded RNA may include modified bases where the modification may be to the phosphate sugar backbone or to the nucleoside. Such modifications may include a nitrogen or sulfur heteroatom or any other modification known in the art.
  • the double-stranded structure may be formed by self-complementary RNA strand such as occurs for a hairpin or a micro RNA or by annealing of two distinct complementary RNA strands.
  • “Overlapping” refers to when two RNA fragments have sequences which overlap by a plurality of nucleotides on one strand, for example, where the plurality of nucleotides (nt) numbers as few as 2-5 nucleotides or by 5-10 nucleotides or more.
  • One or more dsRNAs refers to dsRNAs that differ from each other on the basis of sequence.
  • “Target gene or mRNA” refers to any gene or mRNA of interest. Indeed any of the genes previously identified by genetics or by sequencing may represent a target.
  • Target genes or mRNA may include developmental genes and regulatory genes as well as metabolic or structural genes or genes encoding enzymes.
  • the target gene may be expressed in those cells in which a phenotype is being investigated or in an organism in a manner that directly or indirectly impacts a phenotypic characteristic.
  • the target gene may be endogenous or exogenous.
  • Such cells include any cell in the body of an adult or embryonic animal or plant including gamete or any isolated cell such as occurs in an immortal cell line or primary cell culture.
  • EXAMPLE 1 Production and Characterization of Compositions Comprising a siRNA Complexed With a Polynucleotide Delivery-Enhancing Polypeptide
  • a siRNA Complexed with a Polynucleotide Delivery-Enhancing Polypeptide an adequate amount of siRNA is combined with a pre-determined amount of polynucleotide delivery-enhancing polypeptide, for example in Opti-MEM ® cell medium (Invitrogen), in defined ratios and incubated at room temperature for about 10-30 min.
  • a selected volume, e.g., about 50 ⁇ l, of this mixture is brought into contact with target cells and the cells are incubated for a predetermined incubation period, which in the present example was about 2 hr.
  • the siNA peptide mixture can optionally include cell culture medium or other additives such as fetal bovine serum.
  • cell culture medium or other additives such as fetal bovine serum.
  • Exemplary optimized ratios for transfection efficiency are shown in Table 2 below.
  • Transfections were performed with either regular siRNA or siRNA complexed with one of the above-identified histone proteins on 9L/beta-gal cells.
  • the siRNA was designed to specifically knock down beta-galactosidase mRNA, and activities are expressed as percentage of beta-gal activities from control (control cells were transfected using lipofectamine without the polynucleotide delivery-enhancing polypeptide).
  • Assays for detecting and/or quantifying the efficiency of siRNA delivery are carried out using conventional methods, for example beta-galactosidase assay or flow cytometry methods.
  • 9L/LacZ cells a cell line constitutively expressing beta-galactosidase, were used, and the siRNA against beta-gal mRNA was chemically synthesized and used with delivery reagents to evaluate knock-down efficiency.
  • Transfection Procedure On the first day of the procedure, saturated 9L/LacZ cultures are taken from T75 flasks, and the cells are detached and diluted into 10ml of complete medium (DMEM, lxPS, lxNa Pyruvate, lx NEAA).
  • the cells are further diluted to 1:15, and lOO ⁇ l of this preparation are aliquoted into wells of 96 well plates, which will generally yield about 50% cell confluence by the next day for the transfection. Edges of the wells are left empty and filled with 250 ⁇ l water, and the plates are placed un-stacked in the incubator overnight at 37°C (5% CO 2 incubator). On the second day, the transfection complex is prepared in Opti-MEM, 50 ⁇ l each well. The medium is removed from the plates, and the wells are washed once with 200 ⁇ l PBS or Opti-MEM. The plates are blotted and dried completely with tissue by invertion.
  • the transfection mixture is then added (50 ⁇ l/well) into each well, and 250 ⁇ l water is added to the wells on the edge to prevent them from drying.
  • the cells are then incubated for at least 3 hours at 37°C (5% CO 2 incubator).
  • the transfection mixture is removed and replaced with lOO ⁇ l of complete medium (DMEM, lxPS, lxNa Pyruvate, lx NEAA).
  • the cells are cultured for a defined length of time, and then harvested for the enzyme assay.
  • Enzymatic assay Reagents for the enzymatic assay were purchased from Invitrogen ( ⁇ -Gal Assay Kit, Catalog no.), and Fisher (Pierce Micro BCA Protein Assay Reagent Kit, Catalog).
  • C BCA assay Prepare BSA standard (150ul per well), points should be duplicated on each plate. • Put 145 ⁇ l of water into each well, add 5 ⁇ l of cell lyaste into each well. Prepare final Assay Reagent according to manufacture's instruction. Add 150 ⁇ l of Assay Reagent into each well. Incubate at 37°C for about 20 minutes. Measure the OD at 562nm. D: Calculation of specific activity The specific activity is expressed as nmol of ONPG hydrolyzed/t/mg protein, where t is the time of incubation in minutes at 37°C; mg protein is the protein assayed which is determined by BCA method.
  • Flow Cytometry Measurement of FITC/FAM conjugated siRNA a) After exposure to the complex of siRNA/peptide, cells were incubated for at least 3 hours. b) Wash cells with 200 ⁇ l PBS. c) Detach cells with 15 ⁇ l TE, incubate at 37°C. d) Resuspend cells in five wells with 30 ⁇ l FACS solution (PBS with 0.5% BSA, and 0.1% sodium Azide). e) Combine all five wells into a tube. f) Add PI (Propidium iodide) 5 ⁇ l into each tube. g) Analyze the cells with fluorescence activated cell sorting (FCAS) according to manufacturer's instructions.
  • FCAS fluorescence activated cell sorting
  • siRNA sequence used to silence the beta-galactosidase mRNA was the following: C.U.A.C A.C.A.A.A.U.C A.G.C.G.A.U.U.dT.dT (Sense) (SEQ ID NO:) A.A.AU.C.G.C.U.GA.U.U.U.G.U.G.U.A.G.dT.dT (Antisense) (SEQ ID NO:)
  • siRNA peptide/lipids To evaluate the effects of adding a cationic lipid to a siNA/polynucleotide delivery-enhancing polypeptide mixture, complex or conjugate, the above procedures were followed except the lipofectamine (Invitrogen) was added to siNA/polynucleotide delivery formulation in constant concentrations, following manufacturer's instructions (0.2 ⁇ l/ 100 ⁇ l Opti-MEM). To produce the composition comprised of GKINLKALAALAKKIL (SEQ ID
  • siRNA and LIPOFECTIN® (Invitrogen)
  • the siRNA and peptide were mixed together first in Opti-MEM cell culture medium at room temperature, after which LIPOFECTIN ® was added at room temperature to the mixture to form the siRNA/peptide/cationic lipid composition.
  • LIPOFECTIN ® was added at room temperature to the mixture to form the siRNA/peptide/cationic lipid composition.
  • siRNA and LIPOFECTIN ® were mixed together first in Opti-MEM cell culture medium, into this mixture was added the siRNA to form the siRNA peptide/LIPOFECTIN ® composition.
  • siRNA/peptide/cationic lipid composition using GRKXRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO:) or GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 4) it does not matter in which order the components are added together to produce the siRNA peptide/cationic lipid composition.
  • siRNA mellitin LIPOFECTIN ® To produce the siRNA mellitin LIPOFECTIN ® , the siRNA and mellitin were first mixed together in Opti-MEM cell culture medium and then the LIPOFECTIN ® was added to the mixture. To produce the siRNA/histone HI /LIPOFECTIN ® composition, the histone HI and LIPOFECTIN ® were first added together in Opti-MEM cell culture medium thoroughly mixed and then the siRNA was added, thoroughly and mixed with the histone LIPOFECTIN® mixture to form the siRNA/histone HI/ LIPOFECTIN ® composition.
  • exemplary polynucleotide delivery-enhancing polypeptides of the invention can substantially induce or enhance cellular uptake of siNAs, while the addition of an optional cationic lipid to certain siNA/polypeptide mixtures of the invention may substantially improve siNA delivery efficiency.
  • EXAMPLE 2 Production and Characterization of Compositions Comprising a siRNA Comjugated With a TAT-HA Polynucleotide Delivery-Enhancing Polypeptide
  • the present example describes the synthesis and uptake activity of specific peptides covalently conjugated to one strand of a siRNA duplex. These conjugates efficiently deliver siRNA into the cytoplasm and mediate knockdown of desired target genes.
  • Peptide Synthesis Peptides were synthesized by solid-phase Fmoc chemistry on CLEAR-amide resin using a Rainin Symphony synthesizer. Coupling steps were performed using 5 equivalents of HCTU and Fmoc amino acid with an excess of N-methylmorpholine for 40 minutes. Fmoc removal was accomplished by treating the peptide resin with 20% piperidine in DMF for two 10 minutes cycles. Upon completion of the entire peptide, the Fmoc group was removed with piperidine and washed extensively with DMF.
  • Maleimido modified peptides were prepared by coupling 3.0 equivalents of 3- maleimidopropionic acid and HCTU in the presence of 6 equivalents of N- mefhylmorpholine to the N-terminus of the peptide resin. The extent of coupling was monitored by the Kaiser test.
  • the peptides were cleaved from the resin by the addition of 10 mL of TFA containing 2.5% water and 2.5 triisopropyl silane followed by gentle agitation at room temperature for 2 h.
  • the resulting crude peptide was collected by trituration with ether followed by filtration.
  • the crude product was dissolved in Millipore water and lyophilized to dryness.
  • the crude peptide was taken up in 15 mL of water containing 0.05% TFA and 3 mL acetic acid and loaded onto a Zorbax RX-C8 reversed-phase (22 mm ID x 250 mm, 5 ⁇ m particle size) through a 5 mL injection loop at a flow rate of 5 mL/rnin.
  • the purification was accomplished by running a linear AB gradient of 0.1% B/min where solvent A is 0.05% TFA in water and solvent B is 0.05% TFA in acetonitrile.
  • the purified peptides were analyzed by HPLC and ESMS..
  • RNA molecules are prepared using standard solid phase synthesis methods.
  • the peptide and RNA molecules must be functionalized with specific moieties to allow for covalent attachment to each other.
  • the N-terminus is functionalized, for example, with 3-maleimidopropionic acid.
  • other functional groups such as bromo or iodoacetyl moieties will work as well.
  • the RNA molecule the 5' end of the sense strand or 3' end of the antisense strand is functionalized with, for example, a 1-O-dimethoxytrityl-hexyl-disulfide linker according to the following synthetic method.
  • the 5' modified C6SS-oligonucleotide (GCAAGCUGACCCUGAAGUUCAU; 3.467 mg; 0.4582 ⁇ mol) was reduced to the free thiol group with 0.393 mg (3 eq) of tris(2-carboxyethyl)phosphine (TCEP) in 0.3 ml of 0.1 M triethylamine acetate (TEAA) buffer (pH 7.0) at room temperature for 3 h.
  • TEAA triethylamine acetate
  • IM TEAA buffer pH 7 within 20min and 100% C within next 5 min (t r 21.007 min).
  • MALDI mass spectrometric analysis showed that the peak observed for the conjugate (10 585.3 amu) matches the calculated mass. Yield: 0.509 mg, 0.04815 ⁇ mol, 25.2%.
  • the peptide conjugate sense strand and complimentary antisense strand were annealed in 50 mM potassium acetate, 1 mM magnesium acetate and 15 mM HEPES pH 7.4 by heating at 90 °C for 2 min followed by incubation at 37 °C for 1 h.
  • the formation of the double stranded RNA conjugate was confirmed by non denaturing (15%) polyacrylamide gel elctrophoresis and staining with ethidium bromide.
  • siRNA and peptide were diluted in Opti-MEM® media (Invitrogen), then mixed and allowed to complex 5-10 minutes before adding to cells washed with PBS. Final concentration of siRNA was 500nM at each peptide concentration (2-50 ⁇ M). The conjugate, also diluted in Opti-MEM® media, was added to cells at final concentrations ranging from 62.5nM to 500nM. At 500nM concentration, we also combined with 20% FBS just before adding to washed cells. Cells were transfected for 3 hours at 37°C, 5%CO 2 .
  • siRNA uptake was measured by intensity of Cy5 fluorescence and cellular viability assessed by addition of propidium iodide. As shown in Figures 1 and 2, higher uptake and greater mean fluorescence uptake are observed for the conjugate compared to simply complexing the peptide and RNA. This indicates that in certain embodiments it will be desirable to conjugate the polynucleotide delivery-enhancing polypeptide to the siNA molecule.
  • EXAMPLE 3 Screening of siRNA/Delivery Peptide Complexes Demonstrates Efficient Induction of siRNA Uptake in 9L/LacZ Cells by a Diverse Assemblage of Rationally-Designed Polynucleotide Delivery-Enhancing Polypeptides
  • the present example provides additional evidence that a broad and diverse assemblage of rationally-designed polynucleotide delivery-enhancing polypeptides of the invention induce or enhance siRNA uptake when complexed with siRNAs Approximately 10,000 9L/lacZ cells were plated per well in flat-bottom 96-well plates so that they would be ⁇ 50% confluent the next day at the time of transfection.
  • FAM-labeled siRNA and peptides were diluted in Opti-MEM® media (Invitrogen) at 2- fold the final concentration. Equal volumes of siRNA and peptide were mixed and allowed to complex 5-10 minutes at room temperature and then 50 ⁇ L added to cells, previously washed with PBS. Cells were transfected for 3 hours at 37°C, 5%CO 2 . Cells were washed with PBS, treated with trypsin and then analyzed by flow cytometry. siRNA uptake was measured by intensity of FAM fluorescence and cellular viability assessed by addition of propidium iodide. The results of these screening assays are illustrated in Table 4 below.
  • siRNA/Delivery is Enhanced by Polynucleotide Delivery-Enhancing Polypeptides in Murine Cells
  • the present example illustrates induction/enhancement of siRNA uptake by polynucleotide delivery-enhancing polypeptides of the invention in LacZ cells and also in murine primary fibroblasts.
  • the materials and methods used for these experiments are generally the same as described above, except that for the murine experiments 9L/LacZ cells were replaced with mouse tail fibroblasts.
  • Tables 5 and 6 Table 5 Efficiency of siRNA delivery mediated by rationally-designed 5 polynucleotide delivery-enhancing polypeptides in murine fibroblasts
  • EXAMPLE 5 5 siRNA Delivery is Enhanced by Conjugation of the siRNA to Polynucleotide Delivery-Enhancing Polypeptides
  • the present example provides results from screens to evaluate activity of siRNA/polynucleotide delivery-enhancing polypeptide conjugates for inducing or enhancing siRNAuptake in 9L/LacZ culture cell lines and primary fibroblast from mouse 10 tail.
  • the materials and methods employed for these studies are generally the same as described above, except that no siRNA/peptide mixing is required as needed to produce siRNA/peptide complexes.
  • Tables 7 and 8 Table 7 Efficiency of siRNA delivery mediated by rationally-designed 15 polynucleotide delivery-enhancing polypeptides conjugated to siRNAs in LacZ cells
  • siRNA Gene Expression Knock Down is Enhanced by Polynucleotide Delivery-Enhancing Polypeptides Conjugated to siRNA
  • the instant example demonstrates effective knockdown of target gene expression by siRNA/polynucleotide delivery-enhancing polypeptide complexes of the invention.
  • hTNF- ⁇ human tumor necrosis factor- ⁇
  • Healthy human blood was purchased from Golden West Biologicals (CA), the peripheral blood mononuclear cells (PBMC) were purified from the blood using Ficoll- Pague plus (Amersham) gradient. Human monocytes were then purified from the PBMCs fraction using magnetic microbeads from Miltenyi Biotech. Isolated human monocytes were resuspended in IMDM supplemented with 4mM glutamine, 10% FBS, lx non-essential amino acid and lx pen-strep, and stored at 4C until use. In a 96 well flat bottom plate, human monocytes were seeded at lOOK/well/lOO ⁇ l in OptiMEM medium (Invitrogen). Transfection reagent was mixed with siRNA at desired concentration in OptiMEM medium at room temperature for 20min (for
  • RNA measurement branch DNA technology from Genospectra (CA) was used according to manufacturer's specification.
  • CA Genospectra
  • To quantitate RNA level in the cells both house keeping gene (cypB) and target gene (TNF- ⁇ ) mRNA were measured, and the reading for TNF- ⁇ was normalized with cypB to obtain relative luminescence unit.
  • N165 YC-28 1568- GACCAACTGTCACTCATT 1585 The foregoing studies demonstrate that siRNAs targeting TNF- ⁇ expression are effectively delivered in an active state by polynucleotide delivery-enhancing polypeptides of the invention to mediate knockdown of TNF- ⁇ expression in monocytes.
  • Screening and Characterization Figure 1 characterizes an exemplary assay system for screening siRNA candidate sequences for TNF-a knockdown activity.
  • Human monocytes (CD 14+) treated with LPS induce TNF- ⁇ -specific mRNA within approximately 2 hrs, followed by peak levels of TNF- ⁇ protein 2 hrs later.
  • siRNAs were screened for knockdown activity by transfecting monocytes with siRNA candidate sequences using Lipofectamine 2000, treating infected cells with LPS, and measuring TNF- ⁇ mRNA levels approximately 16 hrs later.
  • siRNA sequences were designed and screened for their ability to knockdown TNF- ⁇ mRNA and protein levels in activated human primary monocytes. Activities for a representative set of 27 siRNA sequences ranged from 80% mRNA knockdown to no detectable activity. In general, TNF- ⁇ protein levels were reduced more than mRNA levels, e.g., a 50% knockdown in TNF- ⁇ mRNA (TNF- ⁇ -1) resulted in a 75% reduction in TNF- ⁇ protein level. Dose response curves for selected siRNAs that exhibited knockdown levels from 30 to 60 % were obtained. Calculated IC 50 values were in the 10- 200 pMolar range. While the siRNA sequences evaluated were distributed throughout the TNF- ⁇ transcript, the most potent siRNAs identified were located in two areas: the middle of the coding region and the 3'-UTR.
  • siRNA Gene Expression Knock Down is Enhanced by Polynucleotide Delivery-Enhancing Polypeptides Complexed with siRNA
  • the present example demonstrates knockdown of target gene expression by peptide-siRNA conjugates of the invention.
  • the materials and methods for these studies are the same as those described above, with the exception that no mixing of the siRNA and peptide is required.
  • the knockdown experiments included comparison of siRNA/peptide-mediated knockdown with and without lipofectamine.
  • the transfection reagent used here was lipofectamine.
  • the cells were replated on the 18 th day due to overgrowth.
  • the second trasnfection was performed on the 19 th day post first transfection.
  • the eGFP levels were measured after the transfection.
  • Scramble or nonsense siRNA(Qiagen) was used as a control, along with a GFPI siRNA (GFPI) and a hairpin siRNA (D#21).
  • the knockdown activities were calibrated with scramble siRNA (Qiagen control)
  • siRNA was serially diluted and combined with a fixed amount of PN73 (1.67uM).
  • the PN73 polynucleotide delivery-enhancing polypeptide was complexed with titration amounts of siRNA.
  • PN73 (1.67uM) was complexed with each titration amount of siRNA for 5 min at RT in OptiMEM medium. After complexation, the complex was added to human monocytes for transfection.
  • EXAMPLE 10 Multiple Dosing Protocol to Extend siRNA Knockdown Effect in Mammalian Cells
  • the instant example demonstrates that multiple dosing schedules will effectively extend gene expression knockdown effects in mammalian cells mediated by siNA/polynucleotide delivery-enhancing polypeptide compositions of the invention.
  • the materials and methods employed for these studies are the same as described above, with the exception that repeated transfections were conducted at the times indicated.
  • the scramble siRNA (Qiagen) was utilized for side by side controls.
  • EXAMPLE 11 In Vivo siRNA/Peptide-Mediated TNF- ⁇ Gene Expression Knock Down
  • the present example provides In Vivo studies demonstrating the efficacy of siRNA/polynucleotide delivery-enhancing polypeptide compositions of the invention to mediate systemic delivery and therapeutic gene knockdown by siRNA, effective to modulate target gene expression and modify phenotype of cells in a therapeutic manner.
  • Human NF- ⁇ expressing mice were purchase from the Hellenic Pasture Institute, Greece) at 5weeks old. Mice were administered through i.v.
  • mice 300 ⁇ l saline twice a week (4 mice), with the RA drug Ramicade (5mg/kg) once a week (2 mice), or with N145 siRNA (2mg/kg) mixed with PN73 at 1:5 molar ratio twice a week (2 mice).
  • plasma samples were collected for ELISA testing (R&D Systems, Cat#SSTA00C), and paw scores were taken twice a week as an accepted index of RA disease progression and therapeutic efficacy.
  • Each paw is given a score between 0 and 3, with the highest score of 12, according to the following scoring index.
  • 0 Normal 1: edema or distortion of paw or ankle joints 2 : distortion of paw and ankle j oints 3: ankylosis of wrist or ankle joints.
  • the results of these paw score evaluations are presented graphically in Figure 3.
  • siNAs of the invention for example siNAs targeting human hTNF- ⁇ - specific mRNAs for degradation, offer higher specificity, lower immunogenicity and greater disease modification than current small molecule, soluble receptor, or antibody therapies for RA. More than 50 candidate siRNA sequences were screened that targeted hTNF- ⁇ and yielded single administration knockdowns of 30-85%.
  • RNA uptake by monocytes Over 20 in silico designed peptide complex and or covalent molecules were compared for fluorescent RNA uptake by monocytes and a number were found to have significantly better uptake than Lipofectamine or cholesterol-conjugated siRNA and with ⁇ 10 pM IC 50 values.
  • the peptide-siRNA formulations efficiently knockdown TNF- ⁇ mRNA and protein levels in activated human monocytes in vitro.
  • One exemplary candidate siRNA/delivery peptide formulation was evaluated in two transgenic mouse models of rheumatoid arthritis (RA) constitutively expressing human TNF- ⁇ .
  • RA rheumatoid arthritis
  • siRNA treated animals showed comparable reductions in RA scores, but significantly lower plasma TNF- ⁇ protein levels than infliximab treated animals.
  • target genes for example cytokines such as TNF- ⁇ , that play important roles in pathological states, such as inflammation, provides effective treatments to alleviate or prevent symptoms of disease, as exemplified by RA, in mammalian subjects.
  • Exemplary siRNA peptide compositions employed within the methods and compositions of the invention provide advantages relating to their ability to reduce or eliminate target gene expression, e.g., TNF- ⁇ expression, rather than by complexing with the product of the target gene, e.g., TNF- ⁇ , as in the case of antibodies or soluble receptors. Improving systemic delivery of nucleic acids according to the teachings of the invention provides yet additional advantages for development of siNAs as drugs. Specific challenges in this context include delivery through tissue barriers to a target cell or tissue, maintaining the stability of the siNA, and intracellular delivery - getting siNAs across cell membranes into cells in sufficient quantities to be effective.
  • the present disclosure demonstrates for the first time an effective in vivo delivery system comprising novel peptide-siRNA compositions targeting specific gene expression, such as expression of human TNF- ⁇ , which attenuate disease activity in transgenic animal models predictive of target diseases, as exemplified by studies using murine models of RA.
  • the compositions and methods of the invention effectively inhibit TNF- ⁇ expression in activated monocytes derived from patients with RA.
  • Table 19 provides a diagram of the primary structure of PN73 and its derivatives generated for optimizing rational design of PN73-based polynucleotide delivery-enhancing polypeptides.
  • the parent peptide PN73 was demonstrated above to be an excellent example of polynucleotide delivery-enhancing polypeptides for inducing or enhancing siRNA delivery to cells, hi order to better understand the function- structural activity relationships of this and other polynucleotide delivery-enhancing polypeptides, primary structural studies were performed by characterizing C- and N- terminal function, and activity of conjugates between PN73 and other chemical moieties.
  • PN73 is a peptide from histone 2B, residues 12-48 aa.
  • PN360 is C-terminal deleted version of PN73 (12-35) and PN361 id N-terminal deleted version of PN73 (23-48).
  • PN404 is a version of PN73 in which all of lysines are replaced with arginines as shown below:
  • PN509 is a pegylated PN73 (PEG molecular weight Ik Dalton ) derivative that is pegylated at the N-terminus.
  • Figure 4 provides the results of uptake efficacy and viability studies in mouse fibroblasts for the foregoing PN73 rationally-designed derivative polynucleotide delivery-enhancing polypeptides. The activity changes of modified PN73 in mouse tail fibroblast cells are illustrated. Unlike PN404, PN509 increases uptake without increasing toxicity.
  • PN73 and PN509 show higher activity in primary cells than Lipofectamine (Invitrogen, CA). The uptake measurements were performed using mouse tail fibroblast cells.

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Abstract

La présente invention a trait à des polypeptides assurant une amélioration de polynucléotides additionnés ou complexés avec, ou conjugués à, des acides nucléiques pour l'amélioration de l'administration d'acides nucléiques dans des cellules. Les acides nucléiques transportés sont actifs dans des cellules cibles en tant que petits acides nucléiques inhibiteurs qui réalisent la modulation de l'expression des gènes cibles, par l'intermédiaire au moins partiellement d'ARN interférence (ARNi). Les compositions à base de petits acides nucléiques inhibiteurs /polypeptides et les procédés de l'invention fournissent des outils efficaces pour la modulation de l'expression générique et la modification de phénotype dans des cellules mammaliennes, y compris la modification de phénotype d'une manière permettant l'élimination de symptômes de maladies et de modification de potentiel de maladies dans des cellules cibles ou chez des sujets individuels auxquels les compositions à base de petits acides nucléiques inhibiteurs/polypeptides sont administrés
EP05804803A 2004-05-04 2005-05-04 Compositions et procedes pour l'amelioration de l'administration d'acides nucleiques dans des cellules et pour la modification de l'expression de genes cibles dans des cellules Withdrawn EP1750775A2 (fr)

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