MXPA06012605A - Compositions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells. - Google Patents

Abstract

Polynucleotide delivery-enhancing polypeptides are admixed or complexed with, or conjugated to, nucleic acids for enhancing delivery the nucleic acids into cells. The transported nucleic acids are active in target cells as small inhibitory nucleic acids (siNAs) that modulate expression of target genes, mediated at least in part by RNA interference (RNAi). The siNA/polypeptide compositions and methods of the invention provide effective tools to modulate gene expression and alter phenotype in mammalian cells, including by altering phenotype in a manner that eliminates disease symptoms or alters disease potential in targeted cells or subject individuals to which the siNA/polypeptide compositions are administered.

Description

Expression of target genes to modify a phenotype, such as a disease state or potential, of cells. BACKGROUND OF THE INVENTION The provision of nucleic acids in plant and animal cells has long been an important object of research and development of molecular biology. Recent developments in the areas of gene therapy, antisense therapy and RNA interference therapy (RNAi) have created a need to develop more efficient means to introduce nucleic acids into cells. A diverse set of plasmids and other nucleic acid "vectors" has been developed to deliver large polynucleotide molecules in cells. Typically these vectors incorporate large DNA molecules that comprise intact genes for the purpose of transforming target cells to express a gene of scientific or therapeutic interest. The process by which exogenous nucleic acids are artificially delivered into cells is generally referred to as transfection. The cells can be transfected to absorb 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 have been developed for transducing cells to deliver exogenous RNA or DNA molecules, including virus-mediated transduction, liposomal delivery or cationic lipid, and numerous methods that target the disruption / penetration of biochemical or mechanical membrane (e.g. example, using detergents, microinjection or particle guns) .- RNA interference is a process of silencing of sequence-specific transcriptional gene in cells initiated by a double-stranded polynucleotide (ds), usually a dsRNA, which is homologous to sequence to a portion of a target messenger RNA (mRNA). The introduction of a suitable dsRNA into cells leads to the destruction of endogenous, analogous mRNAs (ie, mRNAs that share substantial sequence identity with the introduced dsRNA). The dsRNA molecules are split by a nuclease of the RNase III family called dimer in short interfering RNAs (siRNAs), which are 19-23 nucleotides (nt) in length. The siRNAs are then incorporated into a multicomponent nuclease complex known as the RNA-induced silencing complex or "RISC". RISC identifies mRNA substrates through their homology to the siRNA, and effects a silencing of gene expression by binding and destroying the target mRNA. RNA interference raises a promising technology to modify the expression of specific genes in animal and plant cells, and therefore it is expected to provide useful tools to treat a wide range of diseases and disorders available for treatment by modifying gene expression endogenous There remains a long felt need in the matter of better tools and methods for delivering .siRNAs and other small inhibitory nucleic acids (siNAs) in cells, particularly in view of the fact that existing techniques for supplying nucleic acids to cells are limited by poor efficiency and / or high toxicity of the supply reagents. There are related needs for improved methods and formulations to deliver siNAs in an effective amount, in an active and enduring state, and utilize non-toxic delivery vehicles, to selected cells, tissues or compartments to mediate the regulation of gene expression in a manner that modify a phenotype or disease state of the target cells. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the peptide-mediated absorption of complex or conjugated siNAs with a polypeptide that enhances the supply of polynucleotides of the invention (SEQ ID NO: 35). Figure 2 further illustrates the peptide-mediated uptake of complex siNAs or conjugates with a polypeptide that enhances the supply of polynucleotides of the invention (SEQ ID NO: 35). Figure 3 shows paw data for mice injected with siRNA / peptide demonstrating delayed RA progression in treated mice comparable to those shown by subjects treated with Ramicade. Figure 4 provides results of the studies of viability and efficiency of absorption in mouse fibroblasts for polypeptides that improves the supply of derived polynucleotides, rationally designated PN73 of the invention. DESCRIPTION OF EXEMPLIFICATION MODIFIES OF THE INVENTION The present invention meets these needs and fulfills additional objects and advantages by providing novel compositions and methods employing a short interfering nucleic acid (siNA), or a precursor thereof, in combination with a polypeptide that improves the polynucleotide supply. The polypeptide that enhances the polynucleotide delivery is an artificial or natural polypeptide selected for its ability to improve intracellular delivery or absorption of polynucleotides, including siNAs and their precursors. Within the new compositions of the invention, siNA can be mixed or supplemented with, or conjugated with, the polypeptide that enhances the delivery of polynucleotides to form a composition that improves the intracellular delivery of siNA compared to the resulting supply of contacting cells target with a pure siNA (ie, siNA without the polypeptide that improves the present supply). In certain embodiments of the invention, the polypeptide that enhances the delivery of polynucleotides is a histone protein, or a fragment of peptide or polypeptide, derivative, analog or conjugate thereof. Within these embodiments, siNA is mixed, 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, eg, one or more of the following histones: histone H1, histone H2A, histone H2B, histone H3 or histone H4, or one or more of the 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. In more detailed embodiments, the mixture of siRNA / histone, complex or conjugate is substantially free of antipathetic compounds. In other detailed embodiments, siNA that is mixed, complexed or conjugated with the histone protein or polypeptide will comprise a double-stranded RNA, eg, a double-stranded RNA having 30 or fewer nucleotides, and is a short interfering RNA ( siRNA). In exemplary embodiments, the polypeptide that enhances the delivery of histone polynucleotides comprises a histone H2B fragment, as exemplified by the polypeptide that enhances the polynucleotide delivery designated PN73, described herein. In still further detailed embodiments, the polypeptide that enhances polynucleotide delivery can be pegylated to improve stability and / or efficacy, particularly in the context of in vivo administration. Within embodiments of the invention, the polypeptide that enhances polynucleotide delivery is rationally selected or designated to comprise an antipathetic amino acid sequence. For example, polypeptides that enhance the delivery of the polynucleotide can be selected, which comprise a plurality of hydrophobic or non-polar amino acid residues that form a hydrophobic sequence domain or motif, linked to a plurality of charged amino acid residues that form a domain or charged sequence motif producing an unfriendly peptide. In other embodiments, the polypeptide that enhances the delivery of the polynucleotide is selected to comprise a protein transduction domain or motif, and a fusogenic peptide domain or motif. A protein transduction domain is a sequence of peptides that is capable of being inserted into and preferably transiting the cell membrane. A fusogenic peptide is a peptide that is capable of destabilizing a lipid membrane, for example, a plasma membrane or a membrane surrounding an endosome, which can be improved at low pH. The fusogenic or dimeric motifs or motifs are found in a wide variety of viral fusion proteins and other proteins, for example, fibroblast growth factor 4 (FGF4). To rationally designate polypeptides that enhance the delivery of the polynucleotide of the invention, a protein transduction domain is employed as a motif that will facilitate entry of the nucleic acid into a cell through the plasma membrane. In certain embodiments, 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 endosome membrane. The destabilization and disruption of the endosome membrane allows the release of the siNA into the cytoplasm where the siNA can associate with a RISC complex and target its target mRNA. Examples of protein transduction domains for optional incorporation into polypeptides that enhance the delivery of the polynucleotide of the invention include: I. Transduction domain of TAT protein (PTD) (SEQ ID NO: 1) KRRQRRR; 2. Penetratin PTD (SEQ ID NO: 2) RQIKIWFQNRRMKWKK; 3. VP22 PTD (SEQ ID NO: 3) DAATATRGRSAASRPTERPRAPARSASRPRRPVD; 4 Kaposi signal sequences | FGF (SEQ ID NO: 4) AAVALLPAVLLALLAP, and SEQ ID NO: 5) AAVLLPVLLPVLLAAP; 5. human ß3 integrin signal sequence (SEQ ID NO: 6) VTVLALGALAGVGVG; 6. fusion sequence gp41 (SEQ ID NO: 7) GALFLGWLGAAGSTMGA; 7. Caiman crocodylus Ig (v) light chain (SEQ ID NO: 8) MGLGLHLLVLAAALQGA; 8. peptide derived from hCT (SEQ ID NO: 9) LGTYTQDFNKFHTFPQTAIGVGAP; 9. Transport (SEQ ID NO: 10) GWTLNSAGYLLKINLKALAALAKKIL; 10. Loligomer (SEQ ID NO: 11) TPPKKKRKVEDPKKKK; II. Arginine peptide (SEQ ID NO: 12) RRRRRRR; and 12. Amphiphilic model peptide (SEQ ID NO: 13) KLALKLALKALKAALKLA. Examples of fusogenic domains of viral fusion peptides for optional incorporation into polypeptides that enhance the delivery of the polynucleotide of the invention include: 1. Influenza HA2 (SEQ ID NO: 14) GLFGAIAGFIENGWEG; 2. Sendai Fl (SEQ ID NO: 15) FFGAVIGTIALGVATA; 3. respiratory syncytial virus Fl (SEQ ID NO: 16) FLGFLLGVGSAIASGV; 4. HIV gp41 (SEQ ID NO: 17) GVFVLGFLGFLATAGS; and 5. Ebola GP2 (SEQ ID NO: 18) GAAIGLA IPYFGPAA. Within still further embodiments of the invention, polypeptides that enhance the delivery of the polynucleotide are provided, which incorporate a DNA binding domain or motif that facilitates the formation of the polypeptide-siNA complex and / or improves the delivery of siNAs within the methods and compositions of the invention. The DNA binding domains in this context include several "zinc indicator" domains as described for DNA binding regulatory proteins and other proteins identified in Table 1, below (see, for example, Simpson et al., J. Biol. Chem. 278: 28011-28018, 2003). Table 1: Zinc indicator motifs of different DNA binding proteins Zinc indicator motif 665 675 685 695 705 715 Spl ACrCP5ÍCH) S EGRGSG DPSKKKQHIC HIQGCG VYG KTSHLRAHL WETGSRPFC Sp2 ACTCPKCKDG EKRS GEQGKKKHVC HIPDOSKTFR KTSLLRAHW Sp3 ACTCPHCKEG GGRGtN LGKKXQHIC HIPGCGKVYG KTSHLRAHLR WSSGSHFFVC Sp4 ACSCEWCREG EGRG5N EfGK DHIC HIEGCGKVYG KTSffl-RAHLR WKTGSRPFIC DrosBtd R TCPKCTNE SGL.PFIV5P DÉRfífiXQKIC HIPGCERLYG KASHLKTHX WHTGS5PFLC DrosSp TCDCPiJCQEA EKLGPAG - HLRKKNIH5C BIPGCGKVYG KTSHLKAHL WETG3RFFVC CeT22C8.5 RCTCPNCK &I KHG DRSSOHTHLC. SVPGCG TYK KTSHLRAHLR KET D2PFVC Y40B1A.4 PQISLKK IF FIFSNFR- GDGK5 IHIC HL-CHKTYG KTSHLRAHLR GKAGKKPFAC Prospect pattern C-x (2,4) -C-x (12) -H-x (3) -H * The table shows a? Indicator of conservative zinc indicator for union of. Double-stranded DNA that is characterized by the motif pattern C- (2, 4) -Cx (12) -Hx (3) -H, which itself can be used to select and designate polypeptides that enhance the supply of the additional polynucleotide according to the invention. ** The sequences shown in Table 1, for Splr Sp2, Sp3, Sp4, DrosBtd, DrosSp, CeT22C8.5, and Y4pBlA.4, are assigned in this SEQ ID NO: s 19, 20, 21, 22, 23 , 24, 25, and 26, respectively. Alternative DNA binding domains useful for constructing polypeptides that enhance the delivery of the polynucleotide of the invention include, for example, portions of the HIV Tat protein sequence (see, Examples, below). Within the exemplary embodiments of the invention described herein, polypeptides that enhance the supply of the jinnolinucleotide can be rationally designated and constructed by combining any of the above structural elements, domains or motifs in a single effective polypeptide to mediate the improved supply of siNAs in target cells. For example, a protein transduction domain of the TAT polypeptide is fused to the 20 N-terminal amino acids of the influenza virus hemagglutinin protein, termed HA2, to produce a polypeptide that enhances the delivery of the polynucleotide eg emplyificant, herein . Several other polypeptide constructs that enhance the delivery of the polynucleotide are provided in the present description, evidencing that the concepts of the invention are widely applied to create and use a diverse assembly of polypeptides that enhance the delivery of the effective polynucleotide to improve the siNA delivery. Still polypeptides that enhance the delivery of the polynucleotide-exemplary polymers within the invention can be selected from the following peptides: WWETWKPFQCRIC RNFSTRQARRNHRRRHR (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. The polypeptides that enhance the polynucleotide delivery that are useful within the compositions and methods herein comprise all? part of the melittin protein sequence. Still other polypeptides that enhance the delivery of the polynucleotide eg emplificatives are identified in the examples below. Any or a combination of these peptides can be selected or combined to produce polypeptide reagents that enhance the delivery of the polynucleotide to induce or facilitate the intracellular delivery of siNAs within the methods and compositions of the invention. In more detailed aspects of the invention, the mixture, complex or conjugate comprising siRNA and a polypeptide that enhances the delivery of the polynucleotide can optionally be combined with (eg, mixed or complexed with) a cationic lipid, such as LIPOFECTIN®. In this context it is unexpectedly disclosed herein that polypeptides that enhance the delivery of the complex polynucleotide or conjugated to a siRNA will only effect sufficient siNA delivery to mediate genetic silencing by RNAi. However, it is further unexpectedly disclosed herein that a siRNA / polypeptide complex or conjugate that enhances polynucleotide delivery will show even greater activity in mediating the siNA delivery and gene silencing when mixed or complexed with a cationic lipid, such as lipofectin. To produce these compositions comprised of a polypeptide that enhances the delivery of the polynucleotide, siRNA and a cationic lipid, siRNA peptide can first be mixed together in a suitable medium such as a cell culture medium, after which the cationic lipid is added to the mix to form a siRNA / delivery peptide / cationic lipid composition. Optionally, the cationic peptide and lipid can be mixed together first in a suitable medium such as a cell culture medium, then the siRNA can be added to form the siRNA / delivery peptide / cationic lipid composition. Examples of unique cationic lipids within these aspects of the invention include N- [1- (2,3-dioleoyloxy) propyl] -N, N, -trimethylammonium chloride, 1,2-bis (oleoyloxy) -3-3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, -dimethyl-l-propanaminium trifluoroacetate, 1, 3-dioleoyloxy-2- (6-carboxyespermil) -propylamide, 5-carboxyesperitiylglycine dioctadecylamide, tetramethyltetrapalmitoyl spermine, tetramethyltetraoleyl spermine, tetramethyltetralauryl spermine, tetramethyltetramiristyl spermine and tetramethyldiolleyl spermine. DOTMA (N- [l- (2,3-dioleoyloxy) propyl] -N,, N-trimethylammonium chloride, DOTAP (1,2-bis (oleoyloxy) -3, 3- (trimethylammonium) propane), DMRIE bromide of (1, 2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium) or DDAB (dimethyldioctadecylammonium bromide). Polyvalent cationic lipids include lipoespermines, specifically DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, -dimethyl-l-propanaminium trifluoroacetate) and DOSPER 1,3-dioleoyloxy-2- (6-carboxyespermil) ) -propylamide, and di- and tetra-spermines of alkyl tetra-methyl, including but not limited to TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMS (tetramethyltetramiristyl spermine) and TMDOS (tetramethyldiioleyl spermine, DOGS (dioctadecyl-amidoglycylspermine (TRANSFECTAM®)) Other useful cationic lipids are described, for example, in U.S. Patent No. 6,733,777; U.S. Patent No. 6,376,248; No. 5,736,392; U.S. Patent No. 5,686,958; U.S. Patent No. 5,334,761; and U.S. Patent No. 5,459,127.

Cationic lipids are optionally combined with non-cationic lipids, particularly neutral lipids, for example lipids such as (dioleoylphosphatidylethanolamine), DPhPE (diphitanoylphosphatidylethanolamine) or cholesterol. A cationic lipid composition composed of a mixture of DOSPA and DOPE 3: 1 (w / w) or a mixture of DOTMA and DOPE 1: 1 (w / w) (LIPOFECTIN®, Invitrogen) are generally useful in transfecting compositions of this invention. Preferred transfection compositions are those that induce substantial transfection of a higher eukaryotic germ cell. In "exemplary" embodiments, the present invention includes compositions comprising a small nucleic acid molecule, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro- RNA (mRNA), or a small hook RNA (shRNA), mixed or complexed with, or 'conjugated to, a polypeptide that enhances the polynucleotide delivery. As used herein, the term "short interfering nucleic acid", "siNA", short interfering RNA, "siRNA", "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule", or "chemically modified short interfering nucleic acid molecule" refers to any nucleic acid molecule capable of inhibiting or downregulating genetic expression or viral replication, for example, by mediating "RNAi" RNA interference or gene silencing in a specific manner of sequence. Within exemplary embodiments, 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 to sub-regulate the expression, or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to (i.e., that is substantially identical in sequence a) the target nucleic acid sequence or portion thereof. "siNA" means a short interfering nucleic acid, for example, a siRNA, which is a short-length double-stranded nucleic acid (or optionally a longer precursor thereof), and which is not unacceptably toxic in target cells. The length of siNAs useful within the invention in certain embodiments will be optimized to a length of about 21 to 23 bp in length. However, there is no particular limitation on the length of useful siNAs, including siRNAs. For example, siNAs can initially be presented to the cells in a precursor form that is substantially different from a final or processed form of the siNA that will exist and will exert the gene silencing activity in the delivery, or after delivery, to the target cell . The precursor forms of siNAs can, for example, include precursor sequence elements that are processed, degraded, modified, or split at or after the delivery time to produce a siNA that is active within the cell to measure gene silencing. Thus, in certain embodiments, useful siNAs within the invention will have a precursor length, for example, of about 100-200 base pairs, 50-100 base pairs, or less than about 50 base pairs, which will produce a processed siNA, active within the target cell. In other embodiments, a useful siNA or siNA precursor will be about 10 to 49 bp, 15 to 35 bp, or about 21 to 30 bp in length. In certain embodiments of the invention, as noted above, polypeptides that enhance the delivery of polynucleotides are used for the delivery of nucleic acid molecules larger than conventional siNAs, including large siNA nucleic acid precursors. For example, the methods and compositions herein can be used to improve the delivery of larger nucleic acids that represent desired "precursors"., wherein the precursor amino acids can unfold or otherwise be processed before, during or after delivery to a target cell to form an active siNA to modulate gene expression within the target cell. For example, a siNA precursor polynucleotide can be selected as a single, circular filament polynucleotide having two or more turn structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises a sequence of nucleotides 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 may processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. In mammalian cells, dsRNAs longer than 30 base pairs can activate dsRNA PKR-dependent kinase and 2'-5'-oligoadenylate synthetase, usually induced by interferon. Activated PKR inhibits general translation by phosphorylation of the eukaryotic start factor of translational factor 2a (eIF2a), while 2'-5'-oligoadenylate synthetase causes degradation of non-specific mRNA through the activation of RNase L. By virtue of its small size (referring particularly to non-precursor forms), usually less than 30 base pairs, and more commonly between about 17-19, 19-21, or 21-23 base pairs, siNAs of the present invention avoid activation of the response of interferon. In contrast to the nonspecific effect of long dsRNA, siRNA can mediate selective gene silencing in the mammalian system. Hook RNAs, with a short loop and 19 to 27 base pairs in the stem, also selectively silence the expression of genes that are homologous to the sequence in the double-filament stem. Mammalian cells can convert small-hook RNA into siRNA to mediate selective gene silencing. RISC mediates the unfolding of single filament RNA having complementary sequence to the antisense strand of the siRNA duplex. The cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex. Studies have shown that siRNA duplexes of 21 nucleotides are more active when they contain two 3 'nucleotide overhangs. In addition, the complete replacement of one or both filaments of siRNA with nucleotides of 2'-deoxy (2'-H) or 2'-O-methyl suppresses the activity of RNAi, while the substitution of the nucleotides of saline siRNA of 3 'terminal with deoxy (2'-H) nucleotides has been reported to be tolerated. Studies have shown that replacement of 3 'overhang segments of a 21-mer siRNA duplex having 2 nucleotide overhangs with deoxyribonucleotides does not have an adverse effect on the activity of AR i. The replacement of 4 nucleotides at each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity. Alternatively, siNAs can be delivered as single or multiple transcription products expressed by a polynucleotide vector encoding single or multiple siNAs and directing their expression within the target cells. In these embodiments, the double-stranded portion of a final transcription product of siRNAs to be expressed within the target cell can be, for example, 15 to 49 bp, 15 to 35 bp, or approximately 21 to 30 bp in length. Within the emplificativas ej modalities, the double-stranded portions of siNAs, in which two filaments are paired, are not limited to nucleotide segments completely in pairs, and may contain portions not in pairs due to inequality (the corresponding nucleotides are not complementary), protuberance (lacking in the corresponding complementary nucleotide in a filament), protrusion, and the like. The non-paired portions may be contained to the extent that they do not interfere with siNA formation. In more detailed embodiments, a "protuberance" may comprise 1 to 2 nucleotides not in pairs, and the double-stranded region of siNAs in which two filaments are matched, may contain from about 1 to 7, or about 1 to 5 protuberances. In addition, portions of "inequality" contained in the double-stranded region of siNAs may be present in numbers of about 1 to 7, or about 1 to 5. More frequent in the case of inequalities, one of the nucleotides is guanine, and the another is uracil. Such an inequality may be attributable, for example, to a mutation from C to T, G to A, or mixtures thereof, into a corresponding DNA encoding sense RNA, but other causes are also contemplated. Further, in the present invention the double-stranded region of siNAs in which two strands are matched, may contain both protrusion and unevenness portions in the approximate numerical ranges specified. The terminal structure of siNAs of the invention can be either a protuberance or a cohesive (leaving) as long as siNA maintains its activity to silence the expression of target genes. The final cohesive (outgoing) structure is not limited only to the projection 30 as reported by others. In contrast, the 5 'leaving structure can be included as long as it is capable of inducing a gene silencing effect such as by RNAi. In addition, the number of salient nucleotides is not limited to reported limits of 2 or 3 nucleotides, but can be any number as long as the salient does not impair the siNA gene silencing activity. For example, the projections 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 damaged double strand portion and that of a pair comprising single strands projecting at both ends. For example, in the case of a 19 bp double-filament RNA with 4 nucleotide protrusions at both ends, the total length is expressed as 23 bp. In addition, since the outgoing sequence may have low specificity to a target gene, it is not necessarily complementary (antisense) or identical (sense) to the target gene sequence. In addition, as long as siNA is able to maintain its gene silencing effect on the target gene, it may contain low molecular weight structure (e.g., natural RNA molecule such as tRNA, rRNA or viral RNA, or an artificial RNA molecule). ), for example, in the outgoing portion at one end. In addition, the terminal structure of the siNAs may have a stem-loop structure in which the ends of one side of the double-stranded nucleic acid are connected by a linker nucleic acid, eg, a linker RNA. The length of the double filament region (stem-spire portion) can be, for example, 15 to 49 bp, often 15 to 35 bp, and more commonly approximately 21 to 30 bp in length. Alternatively, the length of the double filament region which is a final transcription product of siNAs to be expressed in a target cell can be, for example, about 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bp. long. When the linker segments are used, there is no particular limitation on the length of the linker as long as it does not hide the pairing of the stem portion. For example, for stable pairing of the stem portion and deletion of recombination between DNAs encoding this portion, the linker portion may have a trefoil-leaf tRNA structure. Even if the linker has a length that would hide the pairing of the stem portion, it is possible, for example, constructing the linker portion to include introns so that the introns are cut during the processing of a precursor RNA into mature RNA, thus allowing pairing of the stem portion. In the case of a stem-bicyclic siRNA, any end (head or tail) of RNA without loop structure may have a low molecular weight RNA. As described, these low molecular weight RNAs can include a natural RNA molecule, such as tRNA, rRNA or viral RNA, or an artificial RNA molecule. siNA may also comprise a single filament polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (eg, where such a siNA molecule does not require presence within the siNA molecule nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single filament polynucleotide may further comprise a terminal phosphate group, such as 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 31 -diphosphate As used herein, the term "siNA molecule" is not it limits to molecules containing only DNA or RNA that occurs naturally, but also comprises chemically modified and non-nucleotide nucleotides, In certain embodiments, interference nucleic acid molecules short of the invention 21 -hydroxy (2'-OH) content nucleotides lack. In certain embodiments, the short interfering nucleic acids do not require the presence of nucleotides having a 2'-hydroxy group to mediate RNAi and as such, the short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotide (e.g. nucleotides having a 2'-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi, may nevertheless have a linked linker or linkers and other attached or associated groups, portions, or chains containing one or more nucleotides with 2 'groups -0H. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As . is used herein, the term "siNA" means being equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, eg, short interfering RNA (siRNA), double-stranded RNA (dsRNA) , micro-RNA (mRNA), small-grained RNA (shRNA), short-interference oligonucleotide, short-interference nucleic acid, modified short-interference oligonucleotide, chemically modified siRNA, RNA silencing of post-transcriptional gene (ptgsRNA), and others. In other embodiments, siNA molecules for use within the invention may comprise separate sense and antisense regions or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules, or alternatively non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions and / or stacking interactions. "Antisense RNA" is a strand of RNA that has a sequence complementary to a target gene mRNA, and direct to induce RNAi by binding to the target gene mRNA. "Sense RNA" has a sequence complementary to the antisense RNA, and its antisense RNA is reinforced to form siRNA. These sense and antisense RNAs have been synthesized conventionally with an RNA synthesizer. As used herein, the term "RNAi construction" is a generic term used throughout the specification to include small interfering RNAs (siRNAs), hook RNAs, and other RNA species that can be separated 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 that form dsRNAs or hook RNAs in cells, and / or transcripts that can produce siRNAs in vivo. Optionally, siRNA includes single filaments or double strands of siRNA. A hybrid molecule is a double-stranded nucleic acid that has a similar function to siRNA. Instead of a double-stranded RNA moleculeA hybrid consists of a strand of RNA and a strand of DNA. Preferably, the RNA filament is the antisense filament as that which is the filament that binds to the target mRNA. The hybrid created by the hybridization of the strands of RNA and DNA has a hybridized complementary portion and preferably at least one 3 'overhang. siNAs for use within the invention can be assembled from two separate oligonucleotides where one filament is the sense filament and the other is the antisense filament, wherein the filaments, antisense and sense, are self-complementary (i.e., each filament comprises sequence of nucleotides that is complementary to the nucleotide sequence in the other filament; such as where the antisense filament and sense filament form a duplex or double filament structure, for example, wherein the double filament region is approximately 19 base pairs). The antisense filament may comprise a nucleotide sequence that is complementary to the nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense filament may comprise a nucleotide sequence corresponding to the target nucleic acid sequence or a portion of it. Alternatively, siNA can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of siNA are linked via a non-nucleic acid or nucleic acid-based linker (s). Within additional embodiments, siNAs for intracellular delivery according to the methods and compositions of the invention can be a polynucleotide with a duplex, asymmetric duplex, asymmetric hook secondary structure or hook, having self-complementary sense and antisense regions, wherein the 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. Of the same. Non-limiting examples of chemical modifications that can be made in a siNA include without limitation phosphorothioate internucleotide bonds, 2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, nucleotide nucleotides of "universal base", "acyclic" nucleotides, 5-C-methyl nucleotides, and glyceryl terminal and / or incorporation of abasic inverted desoi residue. These chemical modifications, when used in various siNA constructs, are shown to preserve the RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. In a non-limiting example, the introduction of chemically modified nucleotides into nucleic acid molecules provides a powerful tool for overcoming potential limitations of in vivo stability and inherent bioavailability to native RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can allow a lower dose of a particular nucleic acid molecule for a given therapeutic effect since the chemically modified nucleic acid molecules tend to have a longer half-life in serum. In addition, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and / or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced compared to a native nucleic acid molecule, for example, when compared to an RNA nucleic acid molecule, the total activity of the Modified nucleic acid may be greater than that of the native molecule due to improved stability and / or delivery of the molecule. Unlike native unmodified siNA, chemically modified siNA can also minimize the possibility of activating interferon activity in humans. The siNA molecules described herein, the antisense region of a siNA molecule of the invention may comprise a phosphorothioate internucleotide linkage at the 3 'end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region may comprise about one to five phosphorothioate internucleotide linkages at the 5 'end of said antisense region. In any of the modalities of the siNA molecules described herein, the 3 'terminal nucleotide overhangs of a siNA molecule of the invention may comprise ribonucleotides or deoxyribonucleotides that are chemically modified in a nucleic acid sugar, base, or structure. In any of the embodiments of siNA molecules described herein, the 3 'terminal nucleotide overhangs may comprise one or more universally-based ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3 'terminal nucleotide overhangs may comprise one or more acyclic nucleotides. For example, in a non-limiting example, the invention comprises a small interference chemically modified nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide bonds in a filament siNA. In yet another embodiment, the invention comprises a chemically modified small interference 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 may comprise one or more phosphorothioate internucleotide bonds at the 3 'end, the 5' end, or both of the 3 'and 5' ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention may comprise from about 1 to about 5 or more (eg, to about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide bonds at the 5-terminal end. 'of the felt filament, the antisense filament, or both filaments. In another non-limiting example, an exemplary siNA molecule of the invention may comprise one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) links of pyrimidine phosphorothioate internucleotide in the sense filament, the antisense filament, or both filaments. In yet another non-limiting example, an exemplary siNA molecule of the invention may comprise one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) linkages of purine phosphorothioate internucleotide in the sense strand, the antisense strand, or both strands. A siNA molecule can be comprised of a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (eg, about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (for example, about 18, 19, 20, 21, 22, or 23) base pairs wherein the circular oligonucleotide forms a bell-shaped structure having approximately 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. For example, a circular siNA molecule of the invention is designed, such that degradation of the loop portions of the siNA molecule in vivo can generate a double stranded siNA molecule with 3 'terminal overhangs, such as protrusions. 3 'terminal nucleotide comprising approximately 2 nucleotides. Modified nucleotides present in siNA molecules, preferably in the antisense strand of the siNA molecules, but also optionally in the sense strands and / or both, antisense and sense, comprise modified nucleotides having properties or characteristics similar to ribonucleotides that occur naturally . For example, the invention comprises siNA molecules including modified nucleotides having a Northern conformation (eg, Northern pseodorotation cycle)., see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense filament of the siNA molecules of the invention, but also optionally in the sense filaments and / or both, antisense and sense, are resistant to degradation of nuclease while at the same time maintaining the ability to mediate RNAi. Non-limiting examples of nucleotides having a northern configuration include fixed nucleic acid (LNA) nucleotides (eg, 2'-0, 4'-C-methylene- (D-ribofuranosyl) nucleotides); 2 '-methoxyethoxy nucleotides (MOE); 2'-methyl-thio-ethyl micleotides, 2'-deoxy-2'-fluoro micleotides, 2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides, and 2'-0- nucleotides methyl The sense filament of a double-stranded siNA molecule may have a terminal cap portion such as an inverted deoxy-basic portion, at the 3 'end, 5' end or both, 3 'and 5' ends of the felt filament. Non-limiting examples of conjugates include conjugates and ligands described in Vargeese et al., US Application Ser. Serial No. 10 / 427,160, filed April 30, 2003, incorporated herein by reference in its entirety, including the drawings. In another embodiment, the conjugate is covalently linked to the chemically modified siNA molecule through a biodegradable linker. In one embodiment, the conjugated molecule is attached to the 3 'end of either the sense strand, the antisense strand, or both strands of the chemically modified siNA molecule. In another embodiment, the conjugated molecule is attached to 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 conjugated molecule binds both to the 3 'end and the 5' end of either the sense strand, the antisense strand, the? Arabos filaments of the chemically modified siNA molecule, or any combination thereof. In one embodiment, a conjugated molecule of the invention comprises a molecule that facilitates the delivery of a chemically modified siNA molecule into a biological system, such as a cell. In another embodiment, the conjugated molecule bound to the chemically modified siNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cell uptake. Examples of specific conjugated molecules contemplated by the present invention that can bind 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. 200401102-96, published June 10, 2004. The type of conjugates used and the degree of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and / or stability of siNA constructs while at the same time they maintain the ability of siNA to mediate the activity of AR i. As such, one skilled in the art can select siNA constructs that are modified with several conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example, in animal models as is generally known in The matter. An additional siNA may be further comprised of a mixed nucleotide, non-nucleotide or nucleotide / non-nucleotide linker that binds the sense region of siNA to the antisense region of siNA. In one embodiment, a nucleotide linker can be a " linker " 2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By "aptamer" or "nucleic acid aptamer" as used herein is meant a nucleic acid molecule that specifically binds to a target molecule wherein the nucleic acid molecule has a sequence comprising a sequence recognized by the molecule objective in its natural establishment. Alternatively, an aptamer can be a nucleic acid molecule that binds to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand binding domain of a protein, thus preventing ligand interaction that occurs naturally with the protein. This is a non-limiting example and those in the art will recognize that other modalities can be easily 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. Mol. Ther. , 2: 100 (2000); usser, J. Biotechnol. , 74: 27 (2000); Hermann and Patel, Science 287: 820 (2000); and Jayasena, Clinical Chemistry, 45: 1628. (1999) A non-nucleotide linker may be comprised of an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polimeric compounds (e.g. polyethylene glycols, such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res., 18: 6353 (1990) 'and Nucleic Acids Res., 15: 3113 (1987); Cload and Schepartz, J. Am. Chem. Soc., 113: 6324 (1991); Richardson and Schepartz, J. Am. Chem. Soc, 113: 5109 (1991); Ma et al., Nucleic Acids Res., 21: 2585 (1993) and Biochemistry 32: 1751 (1993); Durand et al., Nucleic Acids Res., 18: 6353 (1990); McCurdy et al., Nucleosides & Nucleotides, 10: 287 (1991); Jschke et al., Tetrahedron Lett. , 34: 301 (1993); Ono et al., Biochemistry, 30: 9914 (1991); Arnold et al., International Publication No. WO 89/02439; Uman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc, 113: 4000 (1991). A "non-nucleotide" further means any group or compound that can be incorporated into a nucleic acid strand in place of one or more nucleotide units, including any sugar and / or phosphate substitution, and allows the remaining bases to show whether the ezimatic The group or compound may be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thyrnine, for example in the Cl position of the sugar. The synthesis of a siNA molecule of the invention, which can be chemically modified, comprises (a) synthesis of two complementary strands of the siNA molecule; (b) strengthening the two complementary strands together under suitable conditions to obtain a double-stranded siNA molecule. In another embodiment, the synthesis of the two complementary filaments of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, the synthesis of the two complementary strands of the siNA molecule is by solid phase random oligonucleotide synthesis. Oligonucleotides (for example, certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using methods known in the art, see for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al. 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, Pat. of EE.ÜÜ. No. 6,001,311. RNA synthesis, including certain siNA molecules of the invention, follow 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. Supplementary and supplement methods for delivering nucleic acid molecules for use within the invention are described, for example, in Akhtar et al., Trends Cell Bio., 2, 139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., Mol. Membr. Biol.f 16: 129-140 (1999); Hofland and Huang, Handb. Exp. Pharmacol. , 137: 165-192 (1999); and Lee et al., ACS Symp. Ser., 752: 184-192 (2000). Sullivan et al., International PCT Publication No. WO 94/02595, further discloses general methods for delivering enzymatic nucleic acid molecules. These methods can be used to supplement or supplement delivery of virtually any nucleic acid molecule contemplated within the invention. Nucleic acid molecules and polypeptides that enhance the delivery of polynucleotides can be administered to cells by a variety of methods known to those skilled in the art, including, but not limited to, administration within the formulations comprising siNA and polypeptide that improves the polynucleotide supply, or further comprising, one or more additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, regulator, stabilizer, preservative, and the like. In certain embodiments, siNA and / or the polypeptide that enhances the polynucleotide delivery can be encapsulated in liposomes, administered by yontophoresis, or incorporated in other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or protein vectors (see, for example, O'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, a nucleic acid / peptide / carrier combination can be delivered locally by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard syringe and needle 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 compositions of the present invention can be used effectively 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 degree) a disease state or other adverse condition in a patient.

Thus, within the additional embodiments the invention provides pharmaceutical compositions and methods that characterize the presence or administration of one or more polynucleic acid (s), typically one or more siNAs, combined, complexed or conjugated with a polypeptide that improves the polynucleotide supply, optionally formulated with a pharmaceutically acceptable carrier, such as a diluent, stabilizer, regulator and the like. The present invention satisfies additional objects and advantages by providing small interference nucleic acid (siNA) molecules that modulate the expression of genes associated with a particular disease state or other adverse condition in a subject. Typically, siNA will target a gene that is expressed at a high level as a contributing or causal factor associated with the disease state of the subject or adverse condition. In this context, siNA will effectively sub-regulate gene expression at levels that prevent, alleviate, or reduce the severity or recurrence of one or more associated symptoms. Alternatively, for several different disease models where expression of the target gene is not necessarily elevated as a consequence or sequela of disease or other adverse condition, the down-regulation of the target gene will nevertheless result in a therapeutic result by decreasing gene expression (i.e. , to reduce levels of a selected mRNA and / or protein product of the target gene). Alternatively, siNAs of the invention may be targeted to decrease the expression of a gene, which may result in upregulation of a "downstream" gene whose expression is negatively regulated by a product or activity of the target gene. Within exemplary embodiments, the compositions and methods of the invention are useful as therapeutic tools for regulating the expression of tumor necrosis factor (TNF-a) to treat or prevent the symptoms of rheumatoid arthritis (RA). In this context the invention further provides compounds, compositions, and methods useful for modulating the expression and activity of TNF-α by RNA interference (RNAi) using small nucleic acid molecules. In more detailed embodiments, the invention provides small nucleic acid molecules, such as small interference nucleic acid (siNA) molecules, small interfering RNA (siRNA), double-stranded RNA (dsRNA),. microRNA (mRNA), and small-hook RNA (shRNA), and related methods, which are effective in modulating the expression of TNF-α and / or TNF-α genes to prevent or alleviate RA symptoms in mammalian subjects. Within these and related therapeutic methods and compositions, the use of chemically modified siNAs will often improve properties of modified siNAs compared to the properties of native siNA molecules, for example, by providing increased resistance to nuclease degradation in vivo, and / or through improved cellular absorption. As can be readily determined according to the description herein, useful ones having multiple chemical modifications will maintain their AR i activity. The siNA molecules of the present invention thus provide reagents and methods useful for a variety of diagnostic applications, target validation, genomic discovery, genetic engineering and pharmacogenomics. These siNAs of the present invention can be administered in any form, for example, transdermally, or by local injection (eg, local injection at sites of psoriatic plaques to treat psoriasis, or at the joints of patients afflicted with psoriatic arthritis or RA). In more detailed embodiments, the invention provides formulations and methods for administering therapeutically effective amounts of siNAs directed against a TNF-cc mRNA that effectively down-regulate the TNF-cc RNA and thus reduce or prevent one or more inflammatory conditions associated with TNF-. Compositions and comparable methods are provided in which the 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 causative or contributing factor associated with the selected disease condition. The siNA / polypeptide mixtures that enhance the polynucleotide delivery of the invention can be administered together with other standard treatments for an objective disease condition, for example together with effective therapeutic agents against inflammatory diseases, such as RA or psoriasis. Examples of combinatorially useful and effective agents in this context include nonsteroidal anti-inflammatory drugs (NSAIDs), methotrexate, gold compounds, D-penicillamine, antimalarials, sulfasalazine, glucocorticoids, and other TNF-a neutralizing agents such as infliximab and entrace. Negatively charged polynucleotides of the invention (eg, RNA or DNA) can be administered to a patient by a standard means, with or without stabilizers, regulators and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard procedures for liposome formation can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for straight administration, sterile solutions, suspensions for injectable administration, and other compositions known in the art. The present invention also includes pharmaceutically acceptable formulations of the compositions described herein. These formulations include salts of the above compounds, for example, acid addition salts, for example, hydrochloric, hydrobromic, acetic and sulfonic acid salts of benzene. A "pharmacological composition" or "formulation" refers to a composition or formulation in a form suitable for administration, for example, systemic administration, in a cell or patient, including for example a human. The appropriate forms, in part, depend on the use or the way of entry, for example, oral, transdermal or by injection. Such forms should prevent the composition or formulation from reaching a target cell (i.e., a cell to which the nucleic acid negatively charged is desirable to deliver). For example, the pharmacological compositions injected into the bloodstream must be soluble. Other factors are known in the art, and include considerations such as toxicity. By "systemic administration" means in vivo accumulation or systemic absorption of drugs in the bloodstream by distribution throughout the body. Routes of administration that lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary or intramuscular. Each of these administration routes exposes the negatively charged polymers, eg, nucleic acids, to an accessible disease tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug vehicle comprising the compounds of the present invention can potentially locate the drug, for example, in certain types of tissue, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate drug association with the surface of cells, such as lymphocytes and macrophages is also useful. This approach can provide improved delivery of the drug to target cells by taking advantage of the macrophage specificity and immune recognition of lymphocyte from abnormal cells, such as cancer cells. By "pharmaceutically acceptable formulation" is meant a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the present invention at the most suitable physical location for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the present invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can improve the entry of drugs in CNS [Jolliet-Riant and Tillement, Fundam. Clin. Pharmacol. , 13: 16-26 (1999)]; biodegradable polymers, such as poly (DL-lactide-co-glycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, DF et al., Cell Transplant, 8: 47-58 (1999)] (Alkermes, Inc. Cambridge , Mass.), And charged nanoparticles, such as those made of polybutyl cyanoacrylates, which can deliver drugs through the blood brain barrier and can modify neuronal absorption mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23: 941-949, (1999)] Other non-limiting examples of delivery strategies for the nucleic acid molecules of the present invention include material described in Boado et al., J. Pharm. Sci., 87: 1308-1315 (1998); Tiler et al., FEBS. Lett., 421: 280-284 (1999), Pardridge et al., PNAS USA., 92: 5592-5596 (1995), Boado, Adv. Drug Delivery Rev., 15: 73-107 (1995); Herrada et al., Nucleic Acids Res., 26: 4910-4916 (1998), and Tiler et al., PNAS USA., 96: 7053-7058 (19). 99) The present invention also includes compositions prepared for storage or administration, including a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable diluent or carrier. Diluents or vehicles acceptable 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, ed., 1985). For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used. A pharmaceutically effective dose is that the dose required to prevent, inhibit the occurrence of, or treat (alleviate a symptom to some degree, preferably all 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 on the potency of the negatively charged polymer. Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum; Wetting or dispersing 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 aliphatic alcohols long chain, for example, heptadecaethyloxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may 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 may 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. Oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. The sweetening agents and flavoring agents may be added to provide acceptable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid. Dispersible granules or powders suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing agent or humectant, suspending agent and one or more preservatives. The dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Suitable excipients, for example, agents, sweeteners, flavors or colorants may also be present. The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally occurring gums, for example, acacia gum or tragacanth gum, naturally occurring phosphatides, for example, soybean seed, lecithin and partial esters or esters derived from fatty acids and hexitol, anhydrides , for example, sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. Emulsions can also contain • sweetening and flavoring agents. The pharmaceutical compositions can be in the form of an injectable, sterile injectable oil or aqueous suspension. This suspension can be formulated according to the known technique using suitable wetting or dispersing agents and suspending agents mentioned above. The sterile injectable preparation can also be a suspension or sterile injectable solution in a non-toxic parenterally acceptable solvent or diluent, for example as a solution of 1,3-butanediol. Among the solvents and acceptable vehicles, which can be used are water, Ringer's solution and solution. of isotonic sodium chloride. In addition, fixed, sterile oils are conveniently employed as a solvent or suspension medium. For this purpose, any soft fixed oil can be employed including mono or synthetic diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. siNAs can also be administered in the form of suppositories, for example, for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irradiating excipient which 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. These siNAs can be extensively modified to improve stability by modification with nuclease-resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 21 -O-methyl, 2'-H. [For a review see üsman and Cedergren, TIBS 17: 34 (1992); üsman et al., Nucleic Acids Symp. Ser. 31: 163 (1994)]. Constructs of siNA can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography and resuspended in water. Nucleic acid molecules chemically synthesized with modifications (base, sugar and / or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency. See, for example, Eckstein et al., International Publication No. WO 92/07065; Perrault et al., Nature 344: 565 (1990); Pieken et al., Science 253, 314 (1991); üsman and Cedergren, Trends in Biochem. Sci. 17: 334 (1992); Ursman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, Pat. from the USA No. 5,334,711; Gold et al., Pat. from the USA No. 6,300,074. All of the above references describe various chemical modifications that can be made to the base, phosphate and / or sugar portions of the nucleic acid molecules described herein. There are several examples in the field describing modifications of sugar, base and phosphate that. they can be introduced into the nucleic acid molecules with significant improvement in their efficacy and nuclease stability. For example, oligonucleotides are modified to improve stability and / or improve biological activity by modification with nuclease-resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 21 -O- methyl, 2'-0-allyl, 2'-H, base nucleotide modifications. For a review see üsman and Cedergren, TIBS. 17:34 (1992); üsman et al., Nucleic Acids Symp. Ser. 31: 163 (1994); Burgin et al., Biochemistry, 35: 14090 (1996). Sugar modification of nucleic acid molecules has been described extensively in the art. See Eckstein et al., PCT International Publication No. WO 92/07065; Perrault et al. Nature, 344, 565-568 (1990); Pieken et al. Science, 253: 314-317 (1991); üsman and Cedergren, Trends in Biochem. Sci. , 17: 334-339 (1992); üsman et al. PCT International Publication No. WO 93/15187; Sproat, Pat. from the USA No. 5, 334, 711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT Publication No. WO 97/26270; Beigelman et al., Pat. from the USA No. 5,716,824; Usman et al., Pat. from the USA No. 5, 627, 053; Woolf et al. , PCT International Publication No. O 98/13526; Thompson et al., Karpeisky et al., Tetrahedron Lett., 39: 1131 (1998); Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences), 48: 39-55 (1998); Verma and Eckstein, Annu. Rev. Biochem. , 67: 99-134 (1998); and Burlina et al., Bioorg. Med. Chem., 5: 1999-2010 (1997). Such publications describe general methods and strategies for determining the location of incorporation of sugar, base and / or phosphate modifications and the like into nucleic acid molecules without modulating the catalysis. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the present invention provided that the ability of siNA to promote RNAi in cells is not significantly inhibited. While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate and / or 5-methylphosphonate bonds improves stability, excessive modifications may cause some toxicity or reduced activity. Therefore, when nucleic acid molecules are designated, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these bonds should decrease the toxicity, resulting in increased efficiency and higher specificity of these molecules. In one embodiment, the invention comprises modified siNA molecules, with modifications of phosphate structure comprising one or more substitutions of fos orothioate, phosphorodithioate,. methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and / or alkylsilyl. For a review of oligonucleotide structure modifications, see Hunziker and Leumann, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417 (1995), and Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994). Methods for the delivery of nucleic acid molecules are described in Akhtar et al., Trends Cell Bio. , 2; 139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, (1995), Maurer et al., Mol. Membr. Biol., 16: 129-140 (1999); Hofland and Huang, Handb. Exp. Pharmacol., 137: 165-192 (1999); and Lee et al., ACS Symp. Ser., 752: 184-192 (2000). Beigelman et al., Pat. from the USA No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further discloses general methods for delivering nucleic acid molecules. These methods can be used for the delivery of virtually any nucleic acid molecule. The nucleic acid molecules can be administered to cells by a variety of methods known to those skilled in the art, including but not limited to, encapsulation in liposomes, by yontophoresis, 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 Publications Nos. WO 03/47518 and WO 03/46185), poly (lactic acid) co-glycolic acid) (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and U.S. Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or Protein vectors (O 'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, the nucleic acid / vehicle combination is delivered locally by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, either subcutaneously, intramuscularly or intradermally, takes 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 present invention can be used as pharmaceutical agents. The pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some degree, preferably all symptoms) of a disease state and a subject. The term "ligand" refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, which is capable of interacting with another compound, such. as a receiver, either directly or indirectly. The receptor that interacts with a ligand may be present on the surface of a cell or may alternatively be an intercellular receptor. The interaction of the ligand with the receptor can result in a biochemical reaction, or it can simply be an association or physical interaction. By "asymmetric hook" as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion which may comprise neo-nucleotides or non-nucleotides, and a sense region which may comprise fewer nucleotides than the antisense region to the extent that the sense region has sufficient complementary nucleotides for the base pair with the antisense region and forms a loop duplex. For example, an asymmetric hook siNA molecule of the invention may comprise an antisense region having sufficient length to mediate RNAi in a T cell (e.g., about 19 to about 22 (e.g., about 19, 20, 21, or 22 nucleotides). ) and a loop region comprising about 4 to about 8 (e.g., about 4, 5, 6, 1, or 8) nucleotides, and a sense region having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) .nucleotides that are complementary to the antisense region.The asymmetric hook siNA molecule can also comprise a 'terminal 5' phosphate group that can be chemically modified The loop portion of the asymmetric hook siNA molecule can comprise any nucleotide, non-nucleotide, linker molecules, or conjugated molecules as described herein. By "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 sufficient nucleotides complementary to the base pair with the antisense region and form a duplex. For example, an asymmetric duplex siNA molecule of the invention may comprise an antisense region having sufficient length to mediate RNAi in a T cell (e.g., about 19 to about 22 (e.g., about 19, 20, 21, or 22 nucleotides) and a sense region having about 3 to about 18 (for example, about 3, 4, 5, 6, 1, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides that are complementary to the antisense region By "modulating gene expression" it is understood that the expression of an objective gene is supraregulated or sub-regulation, which may include supraregulation or down-regulation of mRNA levels present in a cell, or a translation of mRNA, or synthesis of protein or protein subunits, encoded by the target gene Modulation of gene expression can also be determined by the presence, amount, or activity of one or more protein or subunit of protein The target gene that is supraregulates or sub-regulates, so that the expression, level, or activity of the subject protein or subunit is greater than or less than what is observed in the absence of the modulator (eg, a siRNA). For example, the term "modular" may mean "inhibit" but the use of the word "modular" is not limited to this definition. By "inhibiting", "downregulating" or "reducing" the expression, it is understood that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or subunits of protein, or level or activity of a or more proteins or protein subunits encoded by a target gene, is reduced below that observed in the absence of the nucleic acid molecules (eg, siNA) of the invention. In one embodiment, the inhibition, down-regulation or reduction with a siNA molecule is below that level observed in the presence of an attenuated or inactive molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, a siNA molecule with disordered sequence or with inequalities. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the present invention is greater in the presence of the nucleic acid molecule than in its absence. "Silencing" of the gene refers to a loss of partial or complete function through the objective inhibition of gene expression in a cell and can also be referred to as a "deletion". Depending on the circumstances and the biological problem to be addressed, it may be preferable to partially reduce the gene expression.

Alternatively, it may be desirable to reduce the gene expression as much as possible. The degree of silencing can be determined by methods known in the art, some of which are summarized in International Publication No. WO 99/32619. Depending on the analysis, the quantification of gene expression allows the detection of various amounts of inhibition that may be desired in certain embodiments of the invention, including prophylactic and therapeutic methods, which will be able to eliminate the target gene expression, in terms of mRNA levels or protein or activity levels, for example, equal to or greater than 10%, 30%, 50%, 75% 90%, 95% or 99% baseline (ie, normal) or other control levels, including levels of elevated expression as may be associated with particular disease states or other objective conditions for therapy. The phrase "inhibition of expression of an objective gene" refers to the ability of a siRNA of the invention to initiate the silencing of the gene of the target gene. To examine the degree of gene silencing, samples or assays of the organism of interest or cells in culture expressing a particular construct are compared to the control samples lacking expression of the construct. A relative value of 100% is assigned to the control samples (lacking construction expression). Inhibition of expression of a target gene is achieved when the test value relative to the control is approximately 90%, frequently 50%, and in certain modalities 25-0%. Suitable assays include, for example, protein screening or mRNA levels using techniques known to those of skill in the art such as spot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as assays. - phenotypics known to those skilled in the art. By "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 cells that are themselves subject to siNA delivery. "Subject" therefore refers to an organism, organ, tissue, or cell, including in vitro or ex vivo subjects, of organ, tissue or cell, to which the nucleic acid molecules of the invention can be administered and improved by the polypeptides that enhance the polynucleotide delivery described herein. Exemplary subjects include mammalian individuals or cells, for example, human patients or cells. As used in the present "cell" it is used in its usual biological sense, and does not refer to a complete multicellular organism, for example, it does not specifically refer to a human. The cell can be present in an organism, for example, birds, plants and mammals such as humans, cows, sheep, apes, monkeys, pigs, dogs and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., plant or mammalian cell). The cell can be of germline or somatic origin, totipotent or pluripotent, dividing or not - 4 - dividing The cell can also be derived from or may comprise a gamete or embryo, a germ cell, or a fully differentiated cell. By "vectors" is meant any viral based and / or nucleic acid technique used to deliver a desired nucleic acid. By "understanding" is meant including, but not limited to, anything that follows the word "understanding". In this way, the use of the term "comprising" indicates that the items listed are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant to include, and be limited to, whatever the phrase "consisting of" follows. In this way, the phrase "consisting of" indicates that the items listed are required or are mandatory, and that no other element can be present. By "consisting essentially of" is meant to include any element listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or specific action in the description for the items listed. Thus, the phrase "consisting essentially of" indicates that the elements listed are required or are mandatory, but that other elements are optional and may or may not be present depending on whether or not they affect the activity or action of the items listed. By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group in the 2 'position of a portion. beta. -D-ribo-furanosa. The terms include double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from RNA that occurs naturally by the addition , elimination, substitution and / or alteration of one or more nucleotides. Such alterations may include. addition of non-nucleotide material, such as at the end (s) of siNA or internally, for example, in one or more RNA nucleotides. The nucleotides in the RNA molecules of the present invention can also comprise non-standard nucleotides, such as nucleotides that do not occur naturally or nucleotides or chemically synthesized deoxynucleotides. These altered RNAs can be referred to as naturally occurring RNA analogues or analogues. By "highly conserved sequence region" is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the next or from one biological system to the other. By "sense region" is meant a nucleotide sequence of a siNA molecule having complementarity with an antisense region of a siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology to a target nucleic acid sequence. By "antisense region" is meant a nucleotide sequence of a siNA molecule having complementarity with a target nucleic acid sequence. In addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity with a sense region of a siNA molecule. By "target nucleic acid" is meant any sequence of nucleic acids whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA. By "complementarity" is meant that a nucleic acid can form hydrogen bond (s) with another nucleic acid sequence by either traditional Waton-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, for example, RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83: 9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109: 3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick pair formation) with a second nucleic acid sequence (e.g., 5, 6, 7). , 8, 9, or 10 nucleotides out of a total of 10 nucleotides and the first oligonucleotide that is formed in pair with a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). "Perfectly complementary" is understood to mean that all contiguous residues of a nucleic acid sequence will be bound by hydrogen with the same number of contiguous residues in a nucleic acid sequence. The term "universal base" as used herein refers to base nucleotide 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-nitropyrrola, 4-nitroindole, 5-nitroindole, and 6-nitroindole as know in the matter (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447). The term "acyclic nucleotide" as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (Cl, C2, C3, C4, or C5), are independently or in absent combination of the nucleotide. The term "biodegradable" as used herein, refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation. The "biologically active molecule" as used herein, refers to compounds or molecules that are capable of producing 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 present invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogues thereof. The biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and / or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers. The term "phospholipid" as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid may comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, substituted or unsubstituted aryl groups. By "cap structure" are meant chemical modifications, which have been incorporated in any term of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated herein by reference). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can aid in delivery and / or location within a cell. The cap may be present at the 5 'terminus (5' cap) or at the 3 'terminus (3' cap) or may be present in both terms. In non-limiting examples, layer 5 'includes, but is not limited to, glyceryl, abbasic deoxid residue residue (portion): 4', 5 '-methylene nucleotide; 1- (beta-D-eritrofuranosyl), nucleotide 4'-thio; carbocyclic nucleotide; 1, 5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; treo-pentofuranosyl nucleotide; nucleotide 3 ',' -acrylic acid; 3, 4-dihydroxybutyl nucleotide; 3, 5-dihydroxypentyl acyclic nucleotide, 3'-3'-inverted nucleotide portion; basic portion 3 '-3' -inverted; 3 '-2' -inverted nucleotide portion; abasic portion 3 '-21 -inverted; 1, -butanediol phosphate; 3'-phosphoramidate; hexyl phosphate; aminohexyl phosphate; 3'-phosphate; 3 '-phosphorothioate; phosphorodithioate; or bridging or not of methylphosphonate portion. Non-limiting examples of the 3 'cap include, but are not limited to, glyceryl, deoxidic inverted base residue (portion), nucleotide 41, 51 -methylene; nucleotide 1- (beta-D-ertrofuranosyl); 4'-thio nucleotide, carbocyclic nucleotide; phosphate 51 -amino-alkyl; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; phosphate. 1,2-aminododecyl; hydroxypropyl phosphate; 1.5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; treo-pentofuranosyl nucleotide; nucleotide 3 ',' -acrylic acid; nucleotide 3, 4-dihydroxybutyl; 3, 5-dihydroxypentyl nucleotide, 5'-5 '-inverted nucleotide portion; 5 '-5'-inverted abasic portion; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and / or non-bridging 5'-phosphoramidate, phosphorothioate and / or phosphorodithioate, bridging or - li ¬ no bridging of methylphosphonate t 5'-mercapto portions (for more details see Beaucage and lyer, 1993, Tetrahedron 49, 1925; incorporated for reference herein). By the term "non-nucleotide" is meant any group or compound that can be incorporated into a nucleic acid strand in the place of one or more nucleotide units, including either sugar and / or phosphate substitutions, and allows the remaining bases show 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. By "nucleotide" as used herein is as recognized in the art to include natural (standard) bases, and modified bases well known in the art. Such bases are generally located at the 1 'position of a portion of nucleotide sugar. The nucleotides generally comprise base, sugar and a phosphate group. The nucleotides can be modified or unmodified in sugar, phosphate and / or base portion, (also referred interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others; see, for example, üsman and McSwiggen , supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication No. WO 93/15187; Uhlman &Peyman, supra, all incorporated herein by reference). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al, 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be 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 (eg, 5-methylcytidine ), 5-alkyluridines (e.g., ribotimidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), tipping, and others (Burgin et al., 1996 , Biochemistry, 35, 14090; Uhlman &Peyman, supra). "Modified bases" in this aspect are understood to mean bases of nucleotides other than adenine, guanine, cytosine and uracil in position 1 'or their equivalents. By "target site" is meant a sequence within a target RNA that is "target" for cleavage mediated by a siNA construct containing sequences within its antisense region that are complementary to the target sequence. By "detectable level of cleavage" is meant unfolding of target RNA (and formation of split-product RNAs) to a sufficient degree to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. The production of cleavage products of 1-5% of the target RNA is sufficient to detect above the background for most detection methods. By "biological system" is meant, material, in a purified or unpurified form, from biological sources, including but not limited to sources of human, animal, plant, insect, bacterial, viral or other, wherein the system comprises the components required for RNAi activity. The term "biological system" includes, for example, a cell, tissue, or organism or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used as an in vitro establishment. The term "biodegradable linker" as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designated as a biodegradable linker for connecting a molecule to another molecule, eg, a biologically active molecule to a siNA molecule of the invention or the sense and antisense filaments of a siNA molecule of the invention. The biodegradable linker is designated 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 biodegradable linker molecule based on nucleic acid 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'-0-amino, 2'-C-allyl, 2'-0-allyl, and other modified or 21-modified modified nucleotides. The biodegradable nucleic acid linker molecule can be a longer dimer, trimer, tetramer, or nucleic acid molecule, eg, 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 may comprise a single nucleotide with a phosphorus-based bond, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid structure, nucleic acid sugar, or nucleic acid base modifications. By "abasic" is meant portions of sugar lacking a base or having other chemical groups in place of a base in the 1 'position, see for example Adamic et al., Pat. from the USA No. 5,998,203. By "non-modified nucleoside" is meant one of the bases of adenine, cytosine, guanine, thymine, or uracil bound to the 1 'carbon of .beta. -D-ribo-furanosa.

- - By "modified nucleoside" is meant any nucleotide base that 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 Formulas I-VII and / or other modifications described herein. In connection with 2'-nucleotides modified as described for the present invention, "amino" is understood to mean 21 -NH2 or 21 -0-N¾, which may or may not be modified. such modified groups are described, for example, in Eckstein et al., Pat. from the USA No. 5,672,695 and Matulic-Adamic et al., U.S. Patent No. 6,248,878. The siNA molecules can be compounded with cationic lipids, packed into liposomes, or otherwise delivered to target tissues or cells. The nucleic acid or nucleic acid complexes can be administered locally by direct injection, infusion pump or endoprosthesis, with or without their incorporation into biopolymers. In another embodiment, polyethylene glycol (PEG) can be covalently linked to siNA compounds of the present invention, to the polypeptide that enhances the polynucleotide delivery, or both. PEG attached 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 through a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. "Reversed repetition" refers to a sequence of nucleic acids comprising a sense and antisense element positioned so that they are capable of forming 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-split ribozyme between the two elements of the repeat. The elements of the inverted repeat are of sufficient length to form a double-stranded RNA. Typically, each element of the inverted repeat is about 15 to about 100 nucleotides in length, preferably about 20-30 nucleotides base, preferably about 20-25 nucleotides in length, for example, 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 the form of a double or single strand. The term comprises nucleic acids containing known nucleotide analogs or modified structure residues or bonds, which are synthetic, occur naturally and do not occur naturally, which have binding properties such as the reference nucleic acid, and which are metabolized in a similar way to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-0-methyl ribonucleotides, peptide-nucleic acids (PNAs). "Long double-stranded RNA" refers to any double-stranded RNA having a size greater than about 40 base pairs (bp) for example, longer than 100 bp or more particularly larger than 300 bp. The sequence of a long dsRNA may represent a segment of a mRNA or the complete mRNA. The maximum size of the long dsRNA is not limited in the present. the double-stranded RNA may include modified bases where the modification can be for the sugar structure of phosphate or nucleoside. Such modifications may include a sulfur or nitrogen heteroatom or any other modification known in the art. The double filament structure can be formed by self-complementary RNA strand as it occurs for a hook or a micro RNA or by fusing two different complementary strands of DNA. "Coating" refers to when two RNA fragments have sequences that are coated by a plurality of nucleotides in a strand, for example, where the plurality of nucleotide numbers (nt) as few as 2-5 nucleotides or as 5-10 nucleotides or more.

"One or more dsRNAs" refers to dsRNAs that differ from one another in the sequence base. "Target gene or RNA" refers to any mRNA gene of interest. However, any of the genes previously identified by genetics or by sequencing may represent a goal. Target genes or mRNAs can include developmental genes and regulatory genes as well as structural or metabolic genes or genes encoding enzymes. The target gene can be expressed in those cells in which a phenotype is being investigated or in an organism in a way that directly or indirectly impacts a phenotypic characteristic. The target gene can 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. In this specification and the appended claims, the singular forms of "one", "one" and "the" include plural reference unless the context clearly dictates another form. EXAMPLE 1 Production and Characterization of Compositions Comprising a siRNA Composed with a Polypeptide that Enhances the Delivery of Polynucleotide To form complexes between candidate siRNAs and polypeptides that enhance the supply of polynucleotides of the invention, a suitable amount of siRNA is combined with a prerequisite amount. a polypeptide that improves the polynucleotide delivery, for example in Opti-EM® cellular medium (Invitn), in defined proportions and incubated at room temperature for approximately 10-30 min. Subsequently a selected volume, eg, approximately 50 μ ?, of this mixture is contacted with target cells and the cells are incubated for a predetermined incubation period, which in the present example was approximately 2 hr. The siNA / peptide mixture may optionally include cell culture medium or other additives such as fetal bovine serum. For H3, H4 and H2b, a series of experiments is carried out to compose these polypeptides that improve the supply of polynucleotides with siRNA in different proportions. Generally this starts with a 1: 0.01 to 1:50 ratio of siRNA / histone. To each cavity in a 96-well microconcentration plate, 40 μm siNA is added. Each cavity contained beta-gal cells at 50% confluence. The optimized and emplificatory proportions for transfection efficiency are shown in Table 2 below. Transfections are performed with either regular siRNA or compound siRNA with one of the histone proteins - 8 - identified above in 9L / beta-gal cells. siRNA is designated to specifically eliminate beta-galactosidase mRNA, and the activities are expressed as a percentage of control beta-gal activities (control cells are transfected using lipofectamine without the polypeptide that improves the polynucleotide delivery). Analyzes to detect and / or quantify the efficiency of siRNA delivery are carried out using conventional methods, for example, beta-galactosidase analysis or flow cytometry methods. For analysis of beta-galactosidase, 9L / LacZ cells, a cell line constitutively expressing beta-galactosidase, are used, and siRNA against mRNA beta-gal is chemically synthesized and used with delivery reagents to evaluate elimination efficiency. Transfection Procedure The first day of the procedure, saturated 9L / LacZ cultures are taken from T75 flasks, and the cells are separated and diluted in 10ml of complete medium (DMEM, lxPS, lxNa Piruvato, Ix NEAA). The cells are further diluted at 1:15, and ??? μ? of this preparation are aliquoted into 96-well plate cavities, which will generally produce approximately 50% cell confluence by the next day for transfection. The edges of the cavities are allowed to empty and fill with 250 μ? water, and plates are placed unstacked in the incubator overnight at 37 ° C (5% C02 incubator). The second day, the transfection complex is prepared in Opti-MEM, 50μ1 each cavity. The medium is removed from the plates, and the cavities are rinsed once with 200μl PBS or Opti-MEM. The plates are stained and completely dried with fabric by inversion. The transfection mixture is then added (50 μl / well) in each well, and 250 μl water is added to the wells in the rim to prevent them from drying out. The cells are then incubated for at least 3 hours at 37 ° C (5% CO2 incubator). The transfection mixture is removed and replaced with ??? μ? of complete medium (DMEM, lxPS, IxNa Piruvato, lx ????). The cells are cultured for a defined duration of time, and then harvested for enzyme analysis. Enzyme analysis Reagents for enzyme analysis are purchased from Invitrogen (ß-Gal Analysis Kit, Catalog no.), And Fisher (Protein Analysis Reagent Kit, Catalog). A: Cellular lysis • Remove the medium, rinse once with 200μ1 PBS, stain the dry plate with inversion. • Add 30μ1 lysis regulator of ß-Gal equipment in each well. · Freeze-thaw cells twice to generate lysate. B: ß-Gal analysis • Prepare analysis mixture (50μ1 lxregulator, 17μ1 ONPG each cavity) · Take a new plate, add 65μ1 analysis mixture in each cavity. • Add ?? μ? of cell lysate in each cavity. There must be white cavities for subtraction of the previous activities. · Incubate at 37 ° C for approximately 20 minutes, prevent long incubation that will use ONPG and divert high expression. • Add ??? μ? of the Detention solution. • Measure OD at 420nm. C: BCA Analysis • Prepare BSA standard (150ul per cavity), points should be doubled on each plate. • Place 1 5μ1 of water in each cavity, add 5μ1 of cell lysate in each cavity. · Prepare Final Analysis Reagent according to the manufacturer's instructions. • Add 150μ1 of Analysis Reagent in each cavity.

• Incubate at 37 ° C for approximately 20 minutes. • Measure OD at 562nm. D: Specific activity calculation The specific activity is expressed as nmol of hydrolyzed ONPH protein / t / mg, where t is the incubation time in minutes at 37 ° C; mg protein is the analyzed protein that is determined by the BCA method. Measurement of FITC / MAP Flow Cytometry conjugated with siRNA a) After exposure to the siRNA / peptide complex, the cells are incubated for at least 3 hours. b) Rinse the cells with 200μ1 PBS. c) Separate the cells with 15μ1 TE, incubate at 37 ° C. d) Resuspend the cells in five cavities with 30μ1 FACS solution (PBS with 0.5% BSA, and 0.1% Sodium Azide). e) Combine all five cavities in a tube. f) Add PI (propidium iodide) 5μ1 in each tube. g) Analyze the cells with fluorescence activated cell sorting (FCAS) according to the manufacturer's instructions. The siRNA sequence used to silence beta-galactosidase mRNA was as follows: C.Q.ACACAAAÜ.CAGCGAÜ.Ü.U.dT.dT (Sense) (SEQ ID NO: 32) AAAÜ.CGCUGAÜUAGUGUAG dT.dT (Antisense) (SEQ ID NO: 33) Table 2: Efficiency of supply of siRNA mediated by polypeptides that improve the supply of polynucleotides siRNA / peptide / lipids To evaluate the effects of adding a cationic lipid to a siNA / polypeptide mixture that enhances the polynucleotide, complex or conjugate delivery, the above procedures are followed except that lipofectamine (Invitrogen) is added to the delivery formulation of siNA / polynucleotide in constant concentrations, following the manufacturer's instructions (0.2 μ1 / 100 μ? Opti-MEM). To produce the composition comprised of GKINLKALAALAKKIL (SEQ ID NO: 28), siRNA and LIPOFECTIN® (Invitrogen), siRNA and peptide are mixed together first in Opti-MEM cell culture medium at room temperature, after which LIPOFECTIN "is added at room temperature to the mixture to form the siRNA / peptide / cationic lipid composition. producing the composition comprised of RVIRVWFQNKRCKDKK (SEQ ID NO: 29), siRNA and LIPOFECTIN®, the peptide and LIPOFECTIN® are first mixed together in the Opti-MEM cell culture medium, in this mixture siRNA is added to form the siRNA / peptide / LIPOFECTIN® To produce the siRNA / peptide / cationic lipid composition using GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30) or GEQIAQLIAGYIDIILKK KSK (SEQ ID NO: 431) it does not matter in which order the components are added together to produce the siRNA / peptide / cationic lipid composition. To produce siRNA / melittin / LIPOFECTIN®, siRNA and melittin are mixed together first in Opti-MEM cell culture medium and then LIPOFECTIN® is added to the mixture. To produce the siRNA / histone Hl / LIPOFECTIN® composition, histone Hl and LIPOFECTIN® are first added together in Opti-MEM cell culture medium mixed thoroughly and then the siRNA is added, completely and mixed with the histone mixture LIPOFECTIN® to form the siRNA / histone Hl / LIPOFECTIN® composition. Table 3 Efficiency of siRNA supply mediated by polypeptides that improve the supply of polynucleotides with and without cationic lipid Based on the above results, it is apparent that the polypeptides that enhance the polynucleotide delivery of the invention can induce or substantially improve the cellular uptake of siNAs, while the addition of an optional cationic lipid to certain siNA / polypeptide mixtures of the invention can substantially improve the efficiency of siNA supply. EXAMPLE 2 Production and Characterization of Compositions Comprising a conjugated siRNA with a Polypeptide that Enhances the Delivery of TAT-HA Polynucleotides The present example describes the synthesis and absorption of activity of specific peptides covalently conjugated to a strand of a siRNA duplex. These conjugates efficiently deliver siRNA in the cytoplasm and mediate the elimination of desired target genes. Peptide Synthesis Peptides are synthesized by solid phase Fmoc chemistry on CLEAR-amide resin using a Rainin Symphony synthesizer. The coupling steps are performed using 5 equivalents of HCTÜ and amino acid Fmoc with an excess of N-methylforfoline for 40 minutes. Removal of Fmoc is performed by treating the peptide resin with 20% piperidine in DMF for two 10 minute cycles. At the completion of the complete peptide, the Fmoc group is removed with piperidine and rinsed extensively with DMF. The modified maleimido peptides are prepared by coupling 3.0 equivalents of 3-maleimidopropionic acid and HCTU in the presence of 6 equivalents of N-methylmorpholine to the N-terminus of the peptide resin. The degree of coupling is monitored by the Kaiser test. The peptides are cleaved from the resin by the addition of 10 ml of TFA containing 2.5% water and 2.5 triisopropyl silane followed by gentle stirring at room temperature for 2 h. The resulting crude peptide is harvested by trituration with ether followed by filtration. The crude product is dissolved in Millipore water and lyophilized until dry. The crude peptide is absorbed in 15 ml of water containing 0.05% TFA and 3 ml of acetic acid and charged in a reverse phase Zorbax RX-C8 (22 mm ID x 250 m, 5 μm particle size) through an injection loop of 5 ml at a low speed of 5 ml / min. The purification is performed 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 are analyzed by HPLC and ESMS. Synthesis of Conjugates Both peptides and RNA are prepared using standard solid phase synthesis methods. The peptide and RNA molecules must be functionalized with specific portions to allow covalent binding to each other. For the peptide, the N-terminus is functionalized, for example, with 3-maleimidopropionic acid. However, it is recognized that other functional groups such as portions of bromine or iodoacetyl will also work. For the RNA molecule the 5 'end of the sense filament or 3' end of the antisense filament is functionalized with, for example, a 1-O-dimethoxytritylyl hexyl disulphide linker according to the following synthetic method. The modified 5 'C6SS oligonucleotide (GCAAGCUGACCCUGAAGÜÜCAU (SEQ ID NO: 34); 3,467 mg; 0.4582 umol) is 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 regulator (TEAA) (pH 7.0) at room temperature for 3 hours. h. The reduced oligonucleotide is purified by RP HPLC on XTerra® S Ci8 4.6x50mm column using a linear gradient of 0-30% CH3CN in 0.1M TEAA pH 7 regulator within 20min (tr = 5.931 min). Purified reduced oligonucleotide (1.361 mg, 0.19085 μ) was dissolved in 0.2 ml of 0.1 M TEAA regulator pH = 7 and then the peptide with the maleimide portion bound to the peptide N-terminus (0.79 mg, 1.5 g) was add to the oligonucleotide solution. After the addition of peptide, a precipitate immediately formed that disappeared on the addition of 150 μ? of 75% CH3CN / 0.1M TEAA. After stirring overnight at room temperature, the resulting conjugate is purified by RP HPLC on XTerra®MS Ci8 4.6x50mm column using a linear gradient of 0-30% CH3CN in 0.1M TEAA pH 7 regulator within 20min and 100% C within the next 5 min (tr = 21,007 min). The amount of the conjugate is determined spectrophotometrically based on the molar absorption coefficient calculated at λ = 260 nm. Mass spectrometric analysis MALDI showed that the maximum value observed for the conjugate (10 585.3 amu) equals the calculated mass. Production: 0.509 mg, 0.04815 μp ???, 25.2%. The filament of conjugated sense of peptide and complementary antisense filament are reinforced in 50 mM potassium acetate, 1 mM magnesium acetate and 15 mM HEPES pH 7.4 when heated at 90 ° C for 2 min followed by incubation at 37 ° C for 1 h . The formation of the double-stranded RNA conjugate is confirmed by non-denaturing polyacrylamide gel electrophoresis (15%) and staining with ethidium bromide.

Structure of the peptide-siAKN conjugate (SEQ ID NOS 34 and 35) Absorption experiments The cells were placed on the plates the day before in the 24-well plates so that they were ~ 50-80% confluent at the time of transfection. For complexes, siRNA and peptide are diluted in Opti-MEM® medium (Invitrogen), then mixed and allowed to compose 5-10 minutes before adding to cells rinsed with PBS. The final concentration of siRNA was 500nM at each peptide concentration (2-50μ?). The conjugate, also diluted in Opti-MEM® medium, is added to the cells in final concentrations ranging from 62.5nM to 500nM. At a concentration of 500nM, it is also combined with 20% FBS just before adding to runny cells. The cells are transfected for 3 hours at 37 ° C, 5% C02. The cells are rinsed with PBS, treated with trypsin and then analyzed by flow cytometry. The absorption of siRNA is measured by fluorescence intensity Cy5 and cell viability is assessed by the addition of propidium iodide. As shown in Figures 1 and 2, the highest absorption and highest average fluorescence absorption are observed for the conjugate compared to simply composing the peptide and RNA. This indicates that in certain embodiments it will be desirable to conjugate the polypeptide that enhances the supply of polynucleotides to the siNA molecule. EXAMPLE 3 Selection of SIARN / Delivery Peptide Complexes Shows Efficient Induction of SiRNA Absorption in 9L / LacZ Cells by a Miscellaneous Assembly of Polypeptides that Enhance the Provision of Rationally Designated Polynucleotides The present example provides further evidence that a broad and diverse assembly of polypeptides that enhance the supply of rationally designated polynucleotides of the invention induce or enhance the absorption of siRNA when composed with siRNAs. Approximately 10,000 9L / lacZ cells are placed in cavities per cavity in 96-well flat-bottomed plates so that they would be ~ 50% confluent the next day at the time of transfection. siRNA labeled with FAM and peptides are diluted in Opti-MEM® medium (Invitrogen) 2 times the final concentration. Equal volumes of siRNA and peptide are mixed and allowed to make 5-10 minutes at room temperature and then 50 L are added to the cells, previously rinsed with PBS. The cells are transfected for 3 hours at 37 ° C, 5% CO2. The cells are rinsed with PBS, treated with trypsin and then analyzed by flow cytometry. Absorption of siRNA is measured by the intensity of FAM fluorescence and cell viability is assessed by the addition of propidium iodide. The results of these selection tests are illustrated in Table 4 below.

Table 4 Efficiency of siRNA supply mediated by polypeptides that improve the supply of rationally designated polynucleotides% Conc Conc Absorption PN # Sequence Peptide siRNA. (% PI- / FAM +) PN173 GRKKRRQRRRPPQC (SEQ ID NO: 36) LOUM 400nm 84.8 PN227 aleimida- AAVALLPAVLLALLAPRKKRRQRRRPPQ-amide (SEQ ID NO: 37) LUM 400nm AAVALLPAVLLALLAPRKKRRQRRRPPQC 31.0 (SEQ ID PN27 NO: 38) LUM 400nm 82.6 Maleimida- AAVALLPAVLLALLAPRKKRRQRRRPPQ -amide PN275 (SEQ ID NO: 37) 4u 400nM 95.3 NH2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-PN28 amide (SEQ ID NO: 39) 2uM 00nM 79.3 Br c-GRKKRRQRRRPQ-amide (SEQ ID NO: PN69 40) 80uM 400nM 0.0 BrAc- PN81 RRRQRRKRGGDI GEWGNEIFGAIAGFLGamida 8u 800n 97.9 - - (SEQ ID NO: 41) NH2-RRRQRRKRGGDIMGE GNEIFGAIAGFLG-PN250 amide (SEQ ID NO: 35) 15uM 800nM 99.5 PN20 C (YGRK RRQRRRG) 2 (SEQ ID NO: 42) 1.4uM 800nM 82.5 Maleimide-GRKKRRQRRRPP-amide (SEQ ID PN280 NO: 43) 80uM 400nM 7.9 NH2-KLWKAWPKL KKLWKP-amide (SEQ ID PN350 NO: 44) lOuM 400nM 0.0 AAVALLPAVLLALLAPRRRRRR-amide (SEQ ID PN365 NO: 45) lOuM 400nM 81.4 RL RALPRVLRRLLRP-amide (SEQ ID NO: PN366 _46) lOuM 400nM 0.0 NH2-AAVALLPAVLLALLAPSGASGLDKRDYV- PN29 amide (SEQ ID NO: 47) 80u 400nM 86.5 Maleimide- AAVALLPAVLLALLAPSGASGLDKRDYV-amide PN235 (SEQ ID NO: 48) 80u 400nM 0.0 NH2-SGASGLDKRDYVAAVAALLPAVLLALLAP- PN30 amide (SEQ ID NO: 49) 80uM 400nM 0.0 NH2- LLETLL PFQCRICMRNFSTRQARRNHRRRHRR- PN202 amide (SEQ ID NO: 50) 2uM 400nM 70.8 NH2-AAVACRICMRNFSTRQARRNHRRRHRR-amide PN225 (SEQ ID NO: 51) 2uM 400nM 30.9 Maleimide-RQIKI FQNRRMK KK-amide (SEQ PN236 ID NO: 52) lOuM 400nM 37.7 RQIKIWFQNRRM W K amide (SEQ ID NO: PN58 53) 40uM 400nM 75.8 NH2- RQIKIWFQNRRMKWKKDIMGEWGNEIFGAIAGFLG- PN251 amide (SEQ ID NO: 54) 4uM 400nM 44.5 Maleimida- SGRGKQGGKARA AKTRSSRAGLQFPVGRVHRLLRKG PN279 -amide (SEQ ID NO: 55) 40uM 400nM 24.7 SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKG PN61 C-amide (SEQ ID NO: 56) 80uM 800n 86.8 KGSKKAVTKAQKKDGKKRKRSRK-amide (SEQ ID PN360 NO: 57) 80uM 400nM 0.0 NH2-K DGKKRKRSRKESYSVYVYKVLKQ- PN361 amide (SEQ ID NO: 58) lOuM 400nM 42.0 KGSKKAVTKAQK DGKKRKRSR ESYSVYVY VLKQ PN73 (SEQ ID NO: 59) lOuM 400nM 99.5 BrAc-GWTLNSAGILLGKINLKALAALAKKILamide PN64 (SEQ ID NO: 60) lOuM 400nM 14.5 KLALKLALKALKAALKLAamide (SEQ ID NO: PN159 13) .08uM 80nM 16.4 BrAc-KLALKLALKALKAALKLAamida (SEQ ID PN68 NO: 61) lOuM 400nM 0.0 Ac-KETWWETWWTEWSQPKKKRKV-amide (SEQ PN182 ID NO: 62) luM 400nM 84.9 NH2-KETWWETWWTEWSQPGRKKRRQRRRPPQ- PN183 amide (SEQ ID NO: 63) 20uM 400nM 78.1 PN71 BrAc-RRRRRRR (SEQ ID NO: 64) 80uM 400nM 0.0 PN87 QQQQQQQQQ (SEQ ID NO: 65) lOuM 400nM 0.0 NH2-RRRQRRKRGQQQQQQQ-amide (SEQ PN249 ID NO: 66) 80uM 400nM 0.0 PN158 RVIR FQN RC DKK-amide (SEQ ID NO: 67) luM 00nM 94.0 Ac-LGLLLRHLRHHSNLLANI-amide (SEQ ID PN86 NO: 68) 80uM 00nM 62.2 GQMSElEAKVRTVKLARS-amide (SEQ ID NO: PN162 69) 80uM 400nM 0.0 NH2-KL SAWPSLWSSL KP-amide (SEQ ID PN228 NO: 70) 80uM 400nM 6.8 PN357 NH2-KKKKK K-amide (SEQ ID NO: 71) lOuM 400nM 0.0 NH2-AARLHRF NKGKDSTEMRRRR-amide (SEQ PN358 ID NO: 72) 40uM 400nM 0.0 Maleimide-GLGSLLKKAGKKLKQPKSKRKV- PN283 amide (SEQ ID NO: 73) 0uM 400nM 36.3 PN28 Maleimide-Dmt-r-FK-amide (SEQ ID NO:) lOOuM 400nM 0.0 Maleimide-Dmt-r-FKQqQqQqQ-amide PN285 (SEQ ID NO: 74) at 800nM 90.7 PN286 Maleimide- RFK-amide (SEQ ID NO: 75) 80u 400nM 0.0 Maleimide- RFKQqQ + qQqQqQq-amide (SEQ PN289 ID NO: 76) 8uM 400nM 91.7 PN267 Maleimido-YRFK-amide (SEQ ID NO: 77) 80u 00nM 0.3 Maleimide-YRFKYRFKYRFK-amide (SEQ ID PN282 NO: 78) 40uM 800nM 22.8 PN286 Maleimide-WRFK-amide (SEQ ID NO: 75) 80uM 400nM 0.0 Maleimide-WRFKKSKRKV-amide (SEQ ID PN290 NO: 79) 80uM 400nM 5.3 Maleimide-WRFKAAVALLPAVLLALLAP-amide PN291 (SEQ ID NO: 80) 4uM 800nM 12.5 PN243 NH2-Di eYrFKamida (SEQ ID NO: 81) 0uM 400nM 0.0 PN24 NH2-YrFKamide (SEQ ID NO: 82) 80uM 400n 0.0 PN245 NH2-DiMeYRFKamide (SEQ ID NO: 83) 80uM 400nM 0.0 PN2 6 NH2-WrFKamida (SEQ ID NO: 84) 80uM 400nM 0.0 PN247 NH2-DiMeYrW amide (SEQ ID NO: 85) 80uM 400nM 0.0 PN2 8 NH2-KFrDiMeY-amide (SEQ ID NO: 86) 80uM 400nM 0.0 Maleimide- RFKWRFK-amide (SEQ ID NO: PN287 87) lOu 400n 8.8 Maleimide-WRFKWRFKWRFK-amide (SEQ ID PN288 NO: 88) 4u 400nM 9.0 EXAMPLE 4 siRNA / Supply is Enhanced by Improved Polypeptides Supply of Polynucleotides in Murine Cells The present example illustrates induction / enhancement of siRNA absorption by polypeptides that enhance the supply of polynucleotides of the invention in LacZ cells and also in primary murine fibroblasts . The materials and methods used for these experiments are generally the same as described above, except that for murine experiments 9L / LacZ cells are replaced with mouse tail fibroblasts. The results of these studies are provided in Tables 5 and 6 below.

Table 5 Efficiency of siRNA delivery mediated by polypeptides that enhance the supply of rationally designated polynucleotides in murine fibroblasts% Name Sequence State Absorption SiRNA NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG- 0.5uM siRNA / 0uM PN250 araida (SEQ ID NO: 35) peptide 85.9 Cy5 -eGFP NH2- KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVL 0.5u siRNA / 5uM PN 3 KQ-amide (SEQ ID NO: 59) peptide 94.5 Cy5-eGFP Peg-PEG-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVL 0.5uM siRNA / 25uM PN509 KQ-amide (SEQ ID NO: 90) peptide 91 Cy5-eGFP PN404 NH2- 0.5uM siRNA / 25uM 50.4 Cy5-eGFP RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVL RQ-amide peptide (SEQ ID NO: 91) NH2-KKDGKKRKRSRKESYSVYVYKVLKQ- 0.5uM siRNA / 50u PN361 amide (SEQ ID NO: 58) peptide 65 Cy5-eGFP AAVALLPAVLLALLAPRKKRRQRRRPPQC (SEQ 0.5u siRNA / 5u PN27 ID NO: 38) Peptide 60.7 Cy5-eGFP NH2-RQIKIWFQNRRMKWKK-amide (SEQ ID PN58 NO: 53) luM siRNA / 20uM peptide 3.7 Cy5-eGFP NH2-RVIRWFQNKRCKDKK amide (SEQ ID 0.5uM siRNA / 50nM PN158 NO: 67) Peptide 86.2 Cy5-eGFP Maleimido-RVIRWFQNKRSKDKK-amide 0.5uM siRNA / lOOuM PN316 (SEQ ID NO: 92) Peptide 84.8 Cy5-eGFP Maleimide-WRFKQqQqQqQQQq-amide 0.5uM siRNA / 10uM PN289 (SEQ ID NO: 76 peptide 7 Cy5-eGFP NH2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-PN28 amide (SEQ ID NO: 39) luM siRNA / 8u peptide 80.5 Cy5-eGFP 0.5uM siRNA / 130nM PN173 GRKKRRQRRRPPQC (SEQ ID NO: 36) peptide 94.8 Cy5-eGFP KLALKLALKAL AALKLA- amide (SEQ ID 0.5uM siRNA / 5uM PN159 NO: 13) peptide 0 Cy5-eGFP NH2-GWTLNSAGILLGKINLKALAALAKKIL- 0.5uM siRNA / 10nM PN161 amide (SE Q ID NO: 93) peptide 0 Cy5-eGFP Table 6: Efficiency of siRNA supply mediated by polypeptides that improve the supply of rationally designated polynucleotides in LacZ cells and murine fibroblasts % Absorption Cells MTF Cells Peptide Primary lacZ sequence NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-amide PN27 (SEQ ID NO: 94) 86 61 NH2-RK RRQRRRPPQAAVALLPAVLLALLAP-amide PN28 (SEQ ID NO: 89) 79 81 NH2-AAVALLPAVLLALLAPSGASGLD RDYV-amide (SEQ ID NO: 47) PN29 87 not tested PN58 NH2-RQIKIWFQNRRMKWKK-amide (SEQ ID NO: 53) 76 6 PN61 NH2-SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGC-amide 87 not tested (SEQ ID NO: 56) NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ- PN73 amide (SEQ ID NO: 59) 91 95 PN158 NH2 -RVIR FQNKRCKDKK-amide (SEQ ID NO: 67) 94 86 PN173 NH2-GRKKRRQRRRPPQC-amide (SEQ ID NO: 36) 85 95 PN182 NH2-KETWWETWWTEWSQPKKKRKV-amide (SEQ ID NO: 95) 85 not tested NH2-LLETLLKPFQCRIC RNFSTRQARRNHRRRHRR- amide PN202 (SEQ ID NO: 50) 71 not tested PN204 NH2-C (YGRKKRRQRRRG) 2-amide (SEQ ID NO: 42) 83 not tested NH2-RRRQRRKRGGDI GEWGNEIFGAIAGFLG-mida PN250 (SEQ ID NO: 35) 99 86 NH2- KKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: PN361 58) 42 65 PN365 NH2-AAVALLPAVLLALLAPRRRRRR-amide (SEQ ID NO: 45) 81 not tested NH2 -RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ-amide not PN404 (SEQ ID NO: 91) tested 50 NH2-GALFLGFLGAAGST GAWSQPKSKRKVC-amide not PN453 (SEQ ID NO: 96) tested 79 Peg-KGSKKAVT AQKKDG RKRSRKESYSVYVYKVLKQ-amide not PN509 (SEQ ID NO: 90) tested 91 EXAMPLE 5 siRNA / Supply Enhanced by Conjugation of siRNA with Polypeptides Enhancing Polynucleotide Delivery The present example provides selection results to evaluate the activity of siRNA / polypeptide conjugates that improves the supply of polynucleotides to induce or enhance the uptake of siRNA in strains of 9L / LacZ culture cell and primary mouse tail fibroblast. The materials and methods employed for these studies are generally the same as described above, except that no siRNA / peptide mixture is required as necessary to produce siRNA / peptide complexes. The results of these studies are provided in Tables 7 and 8 below. Table 7: Efficiency of siAN delivery mediated by polypeptides that enhance the supply of rationally designated polynucleotides conjugated to siRNAs in LacZ cells% Con ugados siRNA Absorption YRFK (SEQ ID tested CoP267nfR137-l NO: 97) FAM-p-gal up2. OuM 0 WRFK] (SEQ ID FAM-p-gal CoP286nfR138-l NO: 98) 0.8uM 0 (WRFK) 2 (SEQ FAM- -gal CoP287nfR138-l ID NO: 99) 0.8uM 0 FAM-p-gal tested CoP284nfR164 -l Dmt-r-FK up to.OuM 0 (YRFK) 3 (SEQ FAM-p-gal tested CoP282nfR165-l ID NO: 100) up to. OuM 0 WRFKKSKRKV FA -p-gal (SEQ ID NO: tested CoP290nfR165-l 101) up to. OuM 0 CoP277nfR167-l PN73 FAM-p-gal 1. OuM 42.9 CoP277nfR167-2 PN73 FAM-p-gal 2. OuM 55.4 Table 8 Efficiency of siRNA supply mediated by polypeptides that improve the supply of rationally designated polynucleotides conjugated with siRNAs in murine fibroblasts The above data show that a diverse assembly of siRNA / peptide conjugates of the invention mediate the supply of siRNAs in different cell types at high efficiency. EXAMPLE 6 Elimination of Genetic Expression of siRNA is Enhanced by Polypeptides that Enhance the Delivery of Polynucleotides Conjugated with SiRNA The present example demonstrates the effective removal of target gene expression by siRNA / polypeptide complexes that improves the supply of polynucleotides of the invention. In current studies, the ability of the peptide / siRNA complex to modulate the expression of a human tumor necrosis factor-a gene (hTNF-a) gene, implicated in mediating the occurrence or progression of RA when overexpressed in human subjects or other mammals, it is tested. Healthy human blood is purchased from Golden West Biologicals (CA), peripheral blood mononuclear cells (PBMC) are purified from the blood using Ficoll-Pay More (Amersham) gradient. The human monocytes are then purified from the PMBCs fraction using magnetic microbeads from Miltenyi Biotech. Isolated human monocytes are resuspended in IMDM supplemented with 4mM glutamine, 10% FBS, Ix non-essential amino acid and lx pen-strep, and stored at 4C until used. In a 96-well flat bottom plate, the human monocytes are seeded in lOOK / cavity / ??? μ? in the middle - - OptiMEM (Invitrogen). The transfection reagent is mixed with siRNA at a desired concentration in OptiMEM medium at room temperature for 20 min (for Lipofectamine 200; Invitrogen), or 5 min (for peptide). At the end of the incubation, FBS is added to the mixture (final 3%), and 50μl of the mixture is added to the cells. The cells are incubated at 37C for 3 hours. After transfection, the cells are transferred to V-bottom plate, and the cells are formed in pill at 1500rpm / 5min. The cells are resuspended in growth medium (IMDM with glutamine, non-essential amino acid and pen-strep). After incubation overnight, the cells are stimulated with LPS (Sigma) at lng / ml for 3 hours. After induction, cells are harvested as above for quantification of mRNA, and the supernatant is screened for protein quantification. For mRNA measurement, Genospectra branching DNA (CA) technology is used according to the manufacturer's specification. To quantify the level of mRNA in cells, both guard gene mRNA (cypb) and target gene (TNF-a) are measured, and the TNF-a reading is normalized with cypB to obtain relative luminescence unit. To quantify the protein level, TNF-ELISA from BD Bioscience is used according to the manufacturer's specification. siRNAs for these studies are targeted to different target regions of TNF-a mRNA as illustrated in Table 9 below. Table 9 Nomenclature and target sequence for TNF-a target of siRNAs Name Name position Target sequence SEQ ID NO: Alternate N125 TNF-a-1 516-534 GCGTGGAGCTGAGAGATAA. { SEQ ID NO: 109 NI15 TNF-a-2 430-448 GCCTGTAGCCCATGTTGTA 110 N132 TNF-α-3 738-756 GGTATGAGCCCATCTATCT 111 N108 TNF-α- 360-378 CCAGGGACCTCTCTCTAAT 112 N138 TNF-α-5 811-829 GCCCGACTATCTCGACTTT 113 N113 TNF -a-6 424-442 TGACAAGCCTGTAGCCCAT 114 N143 TNF-a-7 844-862 GGTCTACTTTGGGATCATT 115 N107 TNF-a-8 359-377 CCCAGGGACCTCTCTCTAA 116 N137 LC1 806-828 AATCGGCCCGACTATCTCGACTT 117 N122 LC2 514-532 AAUGGCGOGGAGCUGAGAGAU 118 N130 LC3 673-691 AACCUCCDCOCOGCCAUCAAG N101 119 120 N140 1C4 AACUGAAAGCAOGAÜCCGGGA LC5 177-195 820-838 781-799 LC6 AAUCUCGACUUUGCCGAGÜCU N135 121 122 N128 AAGGGÜGACCGACOCAGCGCU LC7 AAUCAGCCGCAÜCGCCGOCOC 636-654 123 612-630 AACCCAUGUGCOCCÜCACCCA LC8 N127 N118 124 472-490 LC9 AAGCOCCAGOGGCÜGAACCGC 125 Nlll LC10 398-416 AAGOCAGAOCAUCÜOCOCGAA 126 N110 LC11 363-381 AAGGGACCÜCUCUCUAAUCAG 127 N105 LC12 265-287 CCTCAGCCTCTTCTCCTTCCTGA 128 N104 LC13 264-282 AAUCCUCAGCCCOUCCUÜ 129 N120 LC14 495-513 AACCAAOGCCCUCCüGGCCAA 130 N153 LC16 1535-1555GTCTAAACAA N136 131 132 N152 CCGACTCAGCGCTGAGATCAA LC17 LC18 787-807 1327-1347 428-448 LC19 CTTGTGATTATTTATTATTTA N114 133 134 N145 AAGCCTGTAGCCCATGTTGTA LC20 982-1002 TAGGGTCGGAACCCAAGCTTA N101 135 177-195 COGAAAGCAUGAUCCGGGA YC-1 YC-2 136 N103 137 251-269 AGGCGGOGCOOGUUCCOCA N106 YC -3 300-318 CCACCACGCUCOUCUGCCO 138 N109 YC-4 362-380 AGGGACCUCUCUCDAAUCA 139 N113 YC-5 424-442 DGACAAGCCOGUAGCCCAU 140 N115 YC-6 430-448 GCCUGUAGCCCAUGDUGÜA 141 N117 YC-7 435-453 OAGCCCAUGOOGÜAGCAAA 142 N120 YC-8 495-513 CCAAÜGCCCUCCDGGCCAA 143 N121 YC-9 510-528 CCAAUGGCGUGGAGCOGAG 144 N123 YC-10 515-533 GGCGÜGGAGCUGAGAGAÜA 145 N125 YC-11 516-534 GCGUGGAGCüGAGAGAÜAA 146 N126 YC-12 558-576 GCCÜGUACCÜCAUCÜACUC 147 N130 YC-13 673-691 CCOCCUCOCüGCCAOCAAG 148 N132 YC- 14 738-756 GGUAUGAGCCCAÜCUAUCO 149 N133 YC-15 772-790 GCUGGAGAAGGGÜGACCGA 150 N134 YC-16 776-794 GAGAAGGGUGACCGACDCA 151 N136 YC-17 787-807 GCCCGACÜAÜCÜCGACUü 152 N141 YC-18 841-859 GCAGGÜCOACÜÜÜGGGAÜC 153 N143 Y C-19 844-862 GGUCUACÜUÜGGGAUCAUU 154 N144 YC-20 853-871 DGGGAUCADUGCCCUGUGA 155 N146 YC-21 985-1003 GGTCGGAACCCAAGCTTAG_156_N147 YC-22 1179-1197 CCAGAATGCTGCAGGACTT 157 N148 YC-23 1198-1216 GAGAAGACCTCACCTAGAA 158 N149 YC-24 1200 1218 GAAGACCTCACCTAGAAAT 159 N150 YC-25 1250-1268 CCAGATGTTTCCAGACTTC 160 N151 YC-26 1312-1330 CTATTTATGTTTGCACTTG 161 N154 YC-27 1547-1565 TCTAAACAATGCTGATTTG 162 N155 YC-28 1568-1585 GACCAACTGTCACTCATT 163 Previous studies demonstrate that siRNAs that target TNF-α expression are effectively delivered in an active state by polypeptides that enhance the polynucleotide delivery of the invention to mediate the elimination of TNF-α expression in monocytes. Table 10 Elimination of TNF-α mediated by a PN73 / siRNA complex Gene Complex target siRNA peptide D (%) TNF-a 4nM 1.6uM TNF-a LC1 PN73 20.08 TNF-a LC2 19.06 TNF-a LC3 23.17 TNF-a LC4 26.67 TNF-a LC5 46.78 TNF-a LC6 44.10 TNF-a LC7 42.76 TNF-a LC8 41.24 TNF-a LC9 40.32 TNF-a LC10 13.52 TNF-a LC11 7.89 TNF-a LC12 40.61 TNF-a LC13 48.29 TNF-a LC14 50.76 TNF-a LC16 55.91 TNF-a LC17 50.78 TNF-a LC18 63.44 TNF-LC19 61.83 TNF-a LC20 42.68 TNF-a YC12 43.60 Table 11 Elimination of TNF-a mediated by complex of PN509 / siRNA 2/24/2005 Obective SiRNA Gene Complex KD peptide (%) TNF-a 4n 1.6uM TNF-a LC1 PN509 31.13 TNF-a LC2 37.04 TNF-a LC3 30.14 TNF-a LC4 22.71 TNF-a LC5 34.93 TNF-a LC6 50.19 TNF-a LC7 56.11 TNF-a LC8 47.35 TNF-a LC9 58.20 TNF-a LC10 25.62 TNF-a LC11 25.65 TNF-a LC12 17.03 TNF-a LC13 25.04 TNF-a LC1 42.78 TNF-a LC16 40.06 TNF-a LC17 48.94 TNF-a LC18 58.13 TNF-a LC19 56.38 TNF-a LC20 71.12 TNF-a YC12 64.37 Table 12 Elimination of TNF-a mediated by a complex PN250 / siAR 2/5/2005 Complex Gene target siRNA peptide KD. { %) TNF-a 20nM PN250 TNF-a YC11 0.5uM 13.70 TNF-a YC12 17.06 TNF-a YC17 17.30 TNF-a YC18 20.72 TNF-a LC13 20.65 T Fa LC20 -3.80 TNF-a TNF-4 0.90 TNF-a YC11 0.75uM 21.09 TNF-a YC12 21.66 TNF-a YC17 29.82 TNF-a YC18 17.82 TNF-a LC13 18.04 TNF-a LC20 10.72 TNF-a TNF-4 14.39 TNF-a YC11 luM 33.10 TNF-a YC12 15.91 TNF-a YC17 24.68 TNF-a YC18 24.66 TNF-a LC13 31.35 TNF-a LC20 26.53 TNF-TNF-4 26.47 The above data show that indeed the levels of elimination of TNF-a gene expression can be achieved in mammalian cells using the new siNA / polypeptide compositions that improves the supply of polynucleotides of the invention. Selection and Characterization Figure 1 characterizes an exemplary analysis system for selecting siRNA candidate sequences for a TNF-a elimination activity. Human monocytes (CD14 +) treated with LPS induce TNF-a specific mRNAs within approximately 2 hrs, followed by maximum TNF-a protein levels after 2 hrs. siRNAs are selected for elimination activity by transfecting monocytes with candidate sequences of siRNA using Lipofectamine 2000, treating cells infected with LPS, and measuring levels of mRNA TNF-about 16 hrs later. Fifty-six siRNA sequences are designated and selected for their ability to eliminate TNF-OI mRNA and protein levels in activated human primary monocytes. Activities for a representative set of 27 siRNA sequences varied from 80% elimination of mRNA to undetectable activity. In general, TNF-α protein levels are reduced more than mRNA levels, for example, a 50% elimination in mRNA TNF-a (TNF-a-1) resulted in a 75% reduction in the level of TNF-a protein. The dose response curves for selected siRNAs that showed elimination levels of 30 to 60% are obtained. The IC50 values calculated were in the range of 10-200 pMolar. While the evaluated siRNA sequences are distributed throughout the transcription of TNF-ci, the more potent siRNAs identified are located in two areas: the middle part of the coding region and 3'-UTR. EXAMPLE 7 Elimination of Genetic Expression of siRNA is Enhanced by Polypeptides that Enhance the Delivery of Polynucleotides Compounds with siRNA The present example demonstrates the removal 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 siRNA and peptide is required. In the present series of studies, elimination experiments included comparison of siRNA / peptide-mediated clearance with or without lipofectamine. Table 13 Elimination mediated by siRNA / peptide expression of T F- with or without lipofectamine cells in Lipofectamine analysis without Lipofectamine cone. conc. peptide siRNA (uM) KD (%) (uM) KD (%) CoP45 6 cIBR LC20 CD1 0 4 no KD 0 4 no KD 1 3 no KD 1 3 no KD 4 no KD 4 no KD CoP45 Peptide 7 T LC20 0 4 no KD 0 4 no KD 1 3 no KD 1 3 no KD 4 no KD 4 no KD CoP27 8 TAT + HA YC12 0 4 no KD 0 4 no KD 1 3 no KD 1 3 no KD 4 no KD 4 no KD CoP27 7 PN73 LC13 MTF 0 19 31 95 0 19 61.61 0 38 32 83 0 38 76.31 0 75 39 29 0 75 73.94 1 50 41 42 1 50 73.14 3 00 39 88 3 00 58.14 6 00 20 23 6 00 50.71 CoP27 7 PN73 LC13 CD14 0 000 93.06 0 002 83.63 0 011 72.58 0 053 73.52 0 266 85.01 CoP27 PN73 LC20 CD14 0 000 75.15 7 0 002 60 72 0 011 57 09 0 053 58 70 0 266 62 79 The above data show that a diverse assembly of polypeptides that enhance the supply of complex polynucleotides of the invention with siRNA function to improve siRNA-mediated clearance of TFN gene expression in mammalian subjects. EXAMPLE 8 Course on Elimination of Genetic Expression siRNA The present example presents studies that relate to the time course of elimination of gene expression mediated by siRNA. To test the duration of the siRNA effect, siRNA transfection procedures as noted above are employed, except that fibroblasts derived from mice expressing eGFP are used. The transfection reagent used here was lipofectamine. Cells are plated on day 18 due to overgrowth. The second transfection is performed on day 19 after the first transfection. On day 19 eGFP levels are measured after transfection. non-sense or disordered siRNA (Qiagen) is used as a control, along with a GFPI siRNA (GFPI) and a hook siRNA (D # 21). - Elimination activities are calibrated with messy siRNA (Qiagen control). Table 14 Course on the Elimination of Genetic Expression of siRNA Previous studies show that siRNA removal activity becomes apparent around day 3, and is sustained until day 9, then target gene expression returns to baseline levels approximately on day 17. After the second transfection the On day 18, another reduction of eGFP expression occurred indicating that the reagent can be repeatedly administered to cells to produce elimination of constant or repeated gene expression. EXAMPLE 9 TNF Expression Elimination Dose Unit SiRNA-mediated Complex with Polypeptide Enhancing Polynucleotide Delivery The present example demonstrates the siRNA-mediated clearance activity with a polypeptide that enhances polynucleotide delivery, PN73, in activated human monocytes is dose-dependent. The SIRAR / PN73 complex is provided in a constant proportion between PN73 and siRNA of approximately PN73: siRNA = 82: 1. 400nM siRNA is complex with 33μ? PN73 for 5min in OptiMEM medium. After the composition, the complex is diluted serially (ratio 1: 2) with OptiMEM. The complex is added to human monocytes for transfection. The next induction and quantification of mRNA is performed according to the above description. Table 15 TNF PN73 Gene Expression Peptide Dosage Unit: siRNA ratio = 82: 1 PN73 siRNA (u) (nM) TNF-2 Control TNF-4 LC8 0 100 100 100 100 1.2 14.81 99.99 80.28 70.22 73.44 3.6 44.44 100.11 69.33 62.97 63.04 11 133.33 99.99 57.82 62.71 59.57 33 400.00 99.99 64.51 78.48 51.30 In a related series of experiments, siRNA are serially diluted and combined with a fixed amount of PN73 (1.67uM). Alternatively established, the polypeptide that enhances the PN73 polynucleotide delivery is complexed with siRNA concentration amounts. PN73 (1.67uM) is complexed with each amount of siRNA concentration for 5 min at RT in OptiMEM medium. After the composition, the complex is added to human monocytes for transfection. The induction and quantification data of mRNA provided in Table 16, below, are obtained by methods described above. Table 16 TNF-cc 1.67uM Genetic Expression Elimination siRNA Dosage Unit PN73 complexed with titration amount of siRNA conc. siRNA (nM) Control LC20 0.8 100.0 84.7 4 100.0 59.4 20 100.0 65.2 100 100.0 54.7 EXAMPLE 10 Multiple Dosing Procedure for Extending the Elimination Effect of siRNA in Mammalian Cells The present example demonstrates that multiple dosing programs will effectively extend the effects of elimination of gene expression in mammalian cells mediated by siNA / polypeptide compositions that improves the supply of polynucleotides of the invention. The materials and methods used for these studies are the same as described above, with the exception that repeated transfections are conducted at the indicated times. Messy siRNA (Qiagen) is used by controls side by side. Table 17 Elimination of Genetic Expression of TNF-a from Repeated siRNA Previous studies show that when multiple transfections are performed on time (in this case between approximately 5-7 days after the first transfection), the effects of deletion of genetic transfection in mammalian cells can be prolonged or re-induced. EXAMPLE 11 Elimination of Genetic Expression of TNF-α Mediated siRNA / In Vivo Peptide The present example provides in vivo studies demonstrating the efficacy of siRNA / polypeptide compositions that improves the polynucleotide delivery of the invention to mediate systemic delivery and elimination of therapeutic gene by siRNA, effective to modulate the objective gene expression and modify the phenotype of cells in a therapeutic manner. Mice expressing human NF-a are purchased from the Hellenic Pasture Institute, Greece) at weeks of age. The mice are administered through i.v. with 300μ1 saline twice a week (4 mice), with the drug RA Ramicade (5mg / kg) once a week (2 mice), or with siRNA N145 (2mg / kg) mixed with PN73 at a molar ratio of 1 : 5 twice a week (2 mice). During the injection periods, plasma samples are collected for ELISA testing (R & D Systems, Cat # SSTA00C), and paw scores are taken twice a week as an accepted rate of RA disease progression and efficacy therapy. Table 18 hT F-a ELISA Age (week) 7 8 9 Ramicade 102.24 39.27 25.80 N145 / PN73 25.96 21.89 14.21 Salina 33.78 34.29 24.48 - 1 - * These data represent the average of the mice in the experiment in pg / ml. The above data demonstrate effective reduction of hTNF- levels in mice treated with siRNA / peptide in circulating blood compared to levels in mice treated with saline (control) or Ramicade. Additional evidence of in vivo efficacy of the siNA / polypeptide compositions that enhance the polynucleotide delivery and methods of the invention are obtained from the above murine subjects using the paw scores, an accepted phenotypic index for RA disease status and treatment efficacy. Due to the difference in the initial point (some animals present with scores in previous points), the scores have been adjusted to 0 for all the animals in the experiments. Each leg is given a score between 0 and 3, with the highest score of 12, according to the following score index. 0: Normal 1: edema or distortion of paw or ankle joints. Distortion of leg and ankle joints. 3: ankylosis of wrist or ankle joints. The results of these paw score evaluations are presented graphically in Figure 3. The data demonstrate that animals injected with siRNA / peptide showed a delayed RA progression that was comparable to that shown by mice treated with Ramicade. The results of the above studies demonstrate that small interfering nucleic acid and polypeptide compositions that enhance the polynucleotide delivery of the invention provide promising new therapeutic tools for regulating gene expression and treating and managing the disease. siNAs of the invention, for example human hTNF- specific mRNAs of siNAs target for degradation, offer higher specificity, lower immunogenicity and greater disease modification than the small current molecule, soluble receptor, or antibody therapies for RA. More than 50 candidate siRNA sequences are selected, which target hTNF-a and produce single administration elimination of 30-85%. Peptide complexes designated in silico and / or covalent molecules are compared for the absorption of fluorescent RNA by monocytes and it is found that a number have significantly better absorption than siRNA conjugated with cholesterol or Lipofectamine and with < 10 pM values IC5o. Peptide-siRNA formulations efficiently remove TNF-a mRNA and protein levels in human monocytes activated in vitro.

An exemplary candidate delivery siRNA / peptide formulation is evaluated in two models of transgenic rheumatoid arthritis (RA) mice constitutively expressing human TNF-α. Animals treated with 2 mg / kg siRNA by IV injection or twice with infliximab twice a week starting at the age of 6 weeks showed stabilization of RA score (inflammation of the leg and joint) beginning at the age of 7 weeks , compared to controls where these disease conditions persisted at week 10. At the age of 9 weeks, animals treated with siRNA showed comparable reductions in RA scores, but plasma TNF-protein levels significantly lower than animals treated with infliximab. Based on the description therein, the use of siRNA to inhibit the expression of target genes, for example, cytokines such as TNF-α, which play important roles in disease states, such as inflammation, provides effective treatments for alleviating or preventing disease symptoms, as exemplified by RA, in mammalian subjects. The siRNA / peptide exemplary compositions employed within the methods and compositions of the invention provide advantages relating to their ability to reduce or eliminate the target gene expression, for example, expression of TNF-a, instead of compounding with the product of the invention. target gene, for example, TNF-OI, as in the case of antibodies or soluble receptors. The improvement of the systemic delivery of nucleic acids according to the teachings of the invention still provides additional advantages for the development of siNAs as drugs. Specific challenges in this context include delivery through tissue barriers to a target cell or tissue, maintaining siNA stability, and intracellular delivery by obtaining siNAs through the cell membrane in 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 that target specific gene expression, such as expression of human TNF-a, which attenuates disease activity in models of transgenic animals predictive of target diseases, as exemplified by studies using RA murine models. In related studies, the compositions and methods of the invention effectively inhibit TNF-α expression in activated monocytes derived from patients with RA. These results indicate that the RNAi pathway effectively mediates the alteration of cell phenotype and disease progression through intracellular effects in TNF trajectories, and avoids the effects of toxicity due to antibody complexes / TNF-a-12- circulating with residual immunoreactivity that characterizes current antibody therapies for RA. Notably, all tests herein are implemented with minimized associated toxicity effects, such that the dosages of siNAs and polypeptides that enhance the polynucleotide delivery shown in these examples always correlate with cell viability levels of at least 80-100%. 90% or more. EXAMPLE 12 Optimized Rational Design of Polypeptides that Improve the Supply of Polynucleotides The present example provides study design and emplificativo data to optimize rational design of polypeptides that improve the supply of polynucleotides of the invention. The manipulations of the subject rational design are conducted for a polypeptide that enhances the histone H2B-derived polynucleotide delivery. Table 19 Removal and modification of PN73 The above table provides a diagram of the structure of PN73 and its derivatives generated to optimize the rational design of polypeptides that improve the supply of polynucleotides based on PN73. The peptide of PN73 origin is demonstrated above to be an excellent example of polypeptides that enhance the delivery of polynucleotides to induce or enhance the delivery of siRNA to cells. To better understand the structural activity-function relationships of this and other polypeptides that enhance the polynucleotide supply, the primary structural studies are carried out by characterizing terminal function C and N, and the activity of conjugates between PN73 and other chemical portions. As noted above, PN73 is a histone 2B peptide, residues 12-48 aa. PN360 is the deleted version of terminal C of PN73 (12-35) and PN361 version deleted of terminal id N of PN73 (23-48). PN404 is a version of PN73 in which all the Usinas are replaced with arginines as shown below: NH2-RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ-amide (SEQ ID NO: 91) PN509 is a pegylated PN73 derivative (PEG lk Dalton molecular weight) which is pegylated in the term N. Figure 4 provides the results of viability studies and absorption efficiency in mouse fibroblasts for polypeptides that enhance the supply of polynucleotide derivatives rationally designated PN73-2 - previous. Changes in modified PN73 activity in mouse tail fibroblast cells are illustrated. Unlike PN404, PN505 increases absorption without increasing toxicity. Although the elimination part of the N term of PN73 reduces activity, the removal of terminal C waste eliminates the activity. Both PN73 and PN509 show higher activity in primary cells than Lipofectamine (Invitrogen, CA). Absorption measurements are made using mouse tail fibroblast cells. Although the above invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the skilled person that certain changes and modifications may be practiced within the scope of the appended claims which are presented by way of illustration without limitation. In this context, several publications and other references have been cited within the above description for economy of description. Each of these references is incorporated herein in its entirety for all purposes. However, it is noted that the various publications discussed herein are incorporated for description only before the filing date of the present application, and the inventors reserve the right to advance such description by virtue of the foregoing invention.

Claims (87)

1. CLAIMS 1. A method for causing the absorption of a double-stranded nucleic acid in an animal cell, comprising incubating said cells with a mixture comprising a polypeptide that enhances the supply of polynucleotides and said nucleic acid.
2. 2. The method of claim 1, wherein the nucleic acid is mixed, complexed or conjugated with the polypeptide that enhances the supply of polynucleotides.
3. 3. The method of claim 1, wherein said nucleic acid is a small inhibitory RNA (siRNA).
4. 4. The method of claim 3, wherein the nucleic acid comprises an siRNA that is complementary to a portion of a TNF- gene.
5. The method of claim 1, wherein the nucleic acid has a length of 30 or less nucleotides or base pairs of nucleotides.
6. The method of claim 1, wherein the polypeptide that enhances the delivery of polynucleotides comprises a histone protein, or a polypeptide or peptide fragment, derivative, analog, or conjugate thereof.
7. The method of claim 1, wherein the polypeptide that improves the polynucleotide delivery comprises an antipathetic amino acid sequence.
8. The method of claim 1, wherein the polypeptide that enhances the polynucleotide delivery comprises a transduction domain of protein or motif.
9. The method of claim 1, wherein the polypeptide that enhances the polynucleotide delivery comprises a fusogenic peptide domain or motif.
10. The method of claim 1, wherein the polypeptide that enhances the polynucleotide delivery comprises a DNA or motif binding domain.
11. The method of claim 1, wherein the polypeptide that enhances the polynucleotide delivery comprises one or more amino acid sequences listed in Tables 2-8 above.
12. The method of claim 1, wherein the polypeptide enhancing the polynucleotide delivery comprises one or more histone proteins selected from histone H1, histone H2B, histone H3, and histone H41, or a fragment thereof, a sequence amino acid selected from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO: 29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and ET KPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4: 1, MW 20,000-50,000), Poly Orn-Trp (4: 1, MW 20,000-50,000), or melitin.
13. The method of claim 1, wherein the polypeptide that improves the supply of polynucleotides is pegylated.
14. The method of claim 1, wherein said mixture further comprises a cationic lipid.
15. 15. The method of claim 14, wherein the cationic lipid is Lipofectin® or Lipofectamine®.
16. 16. A composition comprising a polypeptide that enhances the delivery of polynucleotides and a double-stranded nucleic acid, wherein said composition causes the absorption of said nucleic acid in an animal cell.
17. The composition of claim 16, wherein the nucleic acid is mixed, complexed or conjugated with the polypeptide that enhances the supply of polynucleotides.
18. 18. The composition of claim 16, wherein said nucleic acid is a small inhibitory RNA (siRNA).
19. The composition of claim 18, wherein the nucleic acid comprises a siRNA that is complementary to a portion of a TNF-a gene.
20. The composition of claim 16, wherein the nucleic acid has a length of 30 or less nucleotides or base pairs of nucleotides.
21. The composition of claim 16, wherein the polypeptide that enhances the delivery of polynucleotides comprises a histone protein, or a polypeptide or peptide fragment, derivative, analog, or conjugate thereof.
22. 22. The composition of claim 16, wherein the polypeptide that enhances the polynucleotide delivery comprises an amphipathic amino acid sequence.
23. 23. The composition of claim 16, wherein the polypeptide enhancing the polynucleotide delivery comprises a transduction domain of protein or motif 24.
24. The composition of claim 16, wherein the polypeptide that enhances the polynucleotide delivery comprises a fusogenic peptide domain or motif.
25. The composition of claim 16, wherein the polypeptide that enhances the polynucleotide delivery comprises a DNA binding domain or motif.
26. 26. The composition of claim 16, wherein the polypeptide that enhances the polynucleotide delivery comprises one or more amino acid sequences listed in Tables 2-8 above.
27. The composition of claim 16, wherein the polypeptide enhancing the polynucleotide delivery comprises one or more histone proteins selected from histone H1, histone H2B, histone H3, and histone H41, or a fragment thereof, a sequence amino acid selected from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NOj 29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4: 1, MW 20,000-50,000), Poly Orn-Trp (4: 1, MW 20,000-50,000), or melittin.
28. The composition of claim 16, wherein the polypeptide that improves the supply of polynucleotides is pegylated.
29. 29. The composition of claim 16, further comprising a cationic lipid.
30. 30. The composition of claim 29, wherein the cationic lipid is Lipofectin® or Lipofectamine®.
31. 31. A method for modifying the expression of a target gene in an animal cell, comprising incubating said cell with a mixture comprising a polypeptide that enhances the supply of polynucleotides and a nucleic acid, wherein said nucleic acid is complementary to a region of said target gene.
32. 32. The method of claim 31, wherein the nucleic acid is mixed, complexed or conjugated with the polypeptide that improves the supply of polynucleotides.
33. 33. The method of claim 31, wherein said nucleic acid is a small inhibitory RNA (siRNA).
34. 34. The method of claim 33, wherein the nucleic acid comprises a siRNA that is complementary to a portion of a TNF-a gene.
35. 35. The method of claim 31, wherein the nucleic acid has a length of 30 or less nucleotides or base pairs of nucleotides.
36. 36. The method of claim 31, wherein the polypeptide that enhances the delivery of polynucleotides comprises a histone protein, or a polypeptide or peptide fragment, derivative, analog, or conjugate thereof.
37. 37. The method of claim 31, wherein the polypeptide that enhances the polynucleotide delivery comprises an amphipathic amino acid sequence.
38. 38. The method of claim 31, wherein the polypeptide that enhances the polynucleotide delivery comprises a protein transduction domain or motif
39. 39. The method of claim 31, wherein the polypeptide that enhances the polynucleotide delivery comprises a domain of fusogenic peptide or motif.
40. 40. The method of claim 31, wherein the polypeptide that enhances the polynucleotide delivery comprises a DNA or motif binding domain.
41. 41. The method of claim 31, wherein the polypeptide that enhances the polynucleotide delivery comprises one or more amino acid sequences listed in Tables 2-8 above.
42. 42. The method of claim 31, wherein the polypeptide enhancing the polynucleotide delivery comprises one or more histone proteins selected from histone H1, histone H2B, histone H3, and histone H41, or a fragment thereof, a sequence amino acid selected from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NOj 29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and WWETWKPFQCRIC RNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4: 1, MW 20,000-50,000), Poly Orn-Trp (4: 1, MW 20,000-50,000), or melittin.
43. 43. The method of claim 31, wherein the polypeptide that improves the supply of polynucleotides is pegylated.
44. 44. The method of claim 31, wherein said mixture further comprises a cationic lipid.
45. 45. The method of claim 44, wherein the cationic lipid is Lipofectin® or Lipofectamine®.
46. 46. A composition comprising a polypeptide that enhances the delivery of polynucleotides and a double-stranded nucleic acid, wherein said composition causes the absorption of said nucleic acid in an animal cell, wherein said nucleic acid is complementary to a region of a target gene and modifies the expression of said target gene in said cell.
47. 47. A method for changing a phenotype of an animal subject, comprising administering to said subject a mixture of a polypeptide that enhances the delivery of polynucleotides and a double-stranded nucleic acid, wherein said nucleic acid is complementary to a region of a target gene in said subject.
48. 48. The method of claim 47, wherein said subject is an animal cell or individual.
49. 49. The method of claim 47, wherein the nucleic acid is mixed, complexed or conjugated with the polypeptide that enhances the supply of polynucleotides.
50. 50. The method of claim 47, wherein said nucleic acid is a small inhibitory RNA (siRNA).
51. 51. The method of claim 50, wherein the nucleic acid comprises a siRNA that is complementary to a portion of a TNF-a gene.
52. 52. The method of claim 47, wherein the nucleic acid has a length of 30 or less nucleotides or base pairs of nucleotides.
53. 53. The method of claim 47, wherein the polypeptide that enhances the delivery of polynucleotides comprises a histone protein, or a polypeptide or peptide fragment, derivative, analog, or conjugate thereof.
54. 54. The method of claim 47, wherein the polypeptide that enhances the polynucleotide delivery comprises an amphipathic amino acid sequence.
55. 55. The method of claim 47, wherein the polypeptide that enhances polynucleotide delivery comprises a transduction domain of protein or motif
56. 56. The method of claim 47, wherein the polypeptide that enhances the delivery of polynucleotides comprises a domain. of fusogenic peptide or motif.
57. 57. The method of claim 47, wherein the polypeptide that enhances the polynucleotide delivery comprises a DNA or motif binding domain.
58. 58. The method of claim 47, wherein the polypeptide that enhances the polynucleotide delivery comprises one or more amino acid sequences listed in Tables 2-8 above.
59. 59. The method of claim 47, wherein the polypeptide enhancing the polynucleotide delivery comprises one or more histone proteins selected from histone H1, histone H2B, histone H3, and histone H41, or a fragment thereof, a sequence amino acid selected from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NOj 29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30), GEQ1AQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4: 1, MW 20,000-50,000), Poly Orn-Trp (4: 1, MW 20,000-50,000), or melittin.
60. 60. The method of claim 47, wherein the polypeptide that improves the supply of polynucleotides is pegylated.
61. 61. The method of claim 47, wherein said mixture further comprises a cationic lipid.
62. 62. The method of claim 61, wherein the cationic lipid is Lipofectin® or Lipofectamine®.
63. 63. A mixture comprising a polypeptide that improves the supply of polynucleotides and a double-stranded nucleic acid, wherein said mixture causes the absorption of said nucleic acid in cells of an animal, wherein said nucleic acid is complementary to a region of an objective gene in said cell and is active to modulate the expression of said target gene to mediate a change in the phenotype of the cell or animal.
64. 64. A method for treating an adverse disease or condition in an animal subject comprising administering to said subject an effective amount of a mixture comprising a polypeptide that enhances the supply of polynucleotides and a double-stranded nucleic acid, wherein said mixture causes the absorption of said nucleic acid in cells of the subject, and wherein said nucleic acid is complementary to a region of a target gene in said cells and is active to modulate the expression of said target gene to mediate the prevention or reduction in the occurrence or severity of one or more symptoms of said disease or condition in the subject.
65. 65. The method of claim 64, wherein said subject is an animal cell or individual.
66. 66. The method of claim 64, wherein the nucleic acid is mixed, complexed or conjugated with the polypeptide that enhances the supply of polynucleotides.
67. 67. The method of claim 64, wherein said nucleic acid is a small inhibitory RNA (siRNA).
68. 68. The method of claim 64, wherein the nucleic acid comprises a siRNA that is complementary to a portion of a TNF-a gene.
69. 69. The method of claim 64, wherein the nucleic acid has a length of 30 or less nucleotides or base pairs of nucleotides.
70. 70. The method of claim 64, wherein the polypeptide that enhances the delivery of polynucleotides comprises a histone protein., or a polypeptide or peptide fragment, derivative, analog, or conjugate thereof.
71. 71. The method of claim 64, wherein the polypeptide that enhances the polynucleotide delivery comprises an amphipathic amino acid sequence.
72. 72. The method of claim 64, wherein the polypeptide that enhances polynucleotide delivery comprises a protein transduction domain or motif
73. 73. The method of claim 64, wherein the polypeptide that enhances the polynucleotide delivery comprises a domain. of fusogenic peptide or motif.
74. 74. The method of claim 64, wherein the polypeptide that enhances the polynucleotide delivery comprises a DNA binding domain or motif.
75. 75. The method of claim 64, wherein the polypeptide that enhances the polynucleotide delivery comprises one or more amino acid sequences listed in Tables 2-8 above.
76. 76. The method of claim 64, wherein the polypeptide enhancing the polynucleotide delivery comprises one or more histone proteins selected from histone H1, histone H2B, histone H3, and histone H41, or a fragment thereof, a sequence amino acid selected from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO: 29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and WWET KPFQCRIC RNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4: 1, MW 20,000-50,000), Poly Orn-Trp (4: 1, MW 20,000-50,000), or melittin.
77. 77. The method of claim 64, wherein the polypeptide that improves the supply of polynucleotides is pegylated.
78. 78. The method of claim 64, wherein said mixture further comprises a cationic lipid.
79. 79. The method of claim 78, wherein the cationic lipid is Lipofectin® or Lipofectamine®.
80. 80. A composition comprising a double-stranded nucleic acid (dsNA) having a sense strand and an antisense strand complexed or covalently linked to one or more polypeptides that enhance the supply of polynucleotides.
81. 81. The composition of claim 80, wherein the anti-sense dsNA strand can be hybridized to a mRNA present within a cell of interest.
82. 82. The composition of claim 80, wherein each dsNA strand has 30 or fewer nucleotide pairs.
83. 83. The composition of claim 80, wherein each NA complex of claim 2 wherein each dsNA strand has a length between about 19-25 nucleotides.
84. 84. The composition of claim 80, wherein dsNA comprises a small inhibitory RNA (siRNA).
85. 85. The composition of claim 80, wherein dsNA is a hybrid double stranded nucleic acid. (ds) (Hybrid ds) or has a sense filament and an antisense filament where one of the filaments is a strand of DNA and the other is a strand of RNA.
86. 86. A composition comprising a double-stranded nucleic acid (dsNA) having a sense and an antisense strand mixed, complexed or covalently linked to one or more polypeptides that enhance the delivery of polynucleotides and a cationic lipid.
87. 87. The composition of claim 86, wherein the cationic lipid is selected from the group consisting of N- [1- (2,3-dioleoyloxy) propyl] -N, N-trimethylammonium chloride, 1,2-bis (oleoyloxy) -3-3- (trimethylammonio) propane, 1,2-dimyristyioxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, -dimethyl- l-propanaminium trifluoroacetate, 1,3-dioleoyloxy-2- (6-carboxyespermil) -propylamide, 5-carboxyespermilglycine dioctadecylamide, tetramethyltetrapalmitoyl spermine, tetramethyltetraoleyl spermine, tetramethyltetralauryl spermine, tetramethyltetramiristyl spermine and tetramethyldiioleyl spermine, DOTMA (sodium chloride) N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium), DOTAP (1,2-bis (oleoyloxy) -3,3- (trimethylammonium) propane), DMRIE bromide (1) , 2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium), DDAB (dimethyldioctadecylammonium bromide), polyvalent lipids ca thionics, lipoespermines, DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, -dimethyl-l-propanaminium trifluoroacetate), DOSPER (1,3-dioleoyloxy-2- (6-carboxyespermyl) -propyl) -amid, di- and tetra-spermines of alkyl tetra-methyl, TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethyltetralauryl spermine), TMT S (tetramethyltetramiristyl spermine) TMDOS (tetramethyldiioleyl spermine, DOGS (dioctadecyl-amidoglycylspermine (TRANSFECTAM®), cationic lipids combined with non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DPhPE (difitanoylphosphatidylethanolamine) or cholesterol, a composition of cationic lipids composed of a 3: 1 (w / w) mixture of DOSPA and DOPE, and a 1: 1 (w / w) mixture of DOTMA DOPE.