MX2008003380A - Pharmaceutical compositions for delivery of ribonucleic acid to a cell - Google Patents

Abstract

A composition, method for causing uptake in animal cells of double stranded RNA (dsRNA) and reduction of a target mRNA and a use of a mixture for the production of a medicament for the treatment for Tumor Necrosis Factor-alpha (TNF-α) associated inflammatory condition(s) in an animal subject comprising a polynucleotide delivery-enhancing polypeptide and a dsRNA, wherein the polynucleotide delivery-enhancing polypeptide is amphipathic and comprises nucleic acid binding properties are described.

Description

PHARMACEUTICAL COMPOSITIONS FOR THE SUPPLY OF RIBONUCLEIC ACID TO A CELL TECHNICAL FIELD The invention relates to methods and compositions for the delivery of nucleic acids in cells. More specifically, the invention relates to methods and preparations for the delivery of double-stranded polynucleotides in cells to modify the expression of target genes to alter a phenotype, such as a disease state or potential, of the cells. BACKGROUND OF THE INVENTION The provision of nucleic acids in animal and plant cells has long been an important objective of the research and development of molecular biology. Recent developments in the areas of gene therapy, antisense therapy and RNA interference therapy (RNAi) have created the need to develop more efficient means to introduce nucleic acids into cells. . A diverse array of plasmids and other nucleic acid "vectors" has been developed for the delivery of large polynucleotide molecules in cells. Typically, these vectors incorporate large DNA molecules that comprise intact genes for the purpose of transforming the target cells to express a gene of scientific or therapeutic interest.

The process by which artificially exogenous nucleic acids are delivered to the cells is generally referred to as transfection. The cells can be transfected to absorb a functional nucleic acid from an endogenous 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 to transduce cells to deliver exogenous DNA or RNA molecules, including virus-mediated transduction, cationic or liposomal lipid supply, and numerous methods that target biochemical membrane breakdown / penetration (eg, using detergents, microinjection or particle guns). The interference of. RNA 'is a process of sequence-specific transcriptional gene silencing in cells initiated by a double-stranded polynucleotide (ds), commonly a dsRNA, which is homologous in sequence to a portion of an objectified messenger RNA (mRNA). The introduction of a suitable dsRNA into cells leads to the destruction of endogenous cognate mRNAs (i.e., mRNAs that share a substantial sequence identity with the dsRNA introduced). The dsRNA molecules are split by a nuclease of the NRase III family called dicer 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". The RISC identifies the mRNA substrates through their homology to the siRNA, and effects the silencing of the gene expression by binding and destroying the target mRNA. RNA interference is the emergence of a promising technology to modify the expression of specific genes in plant and animal cells, and consequently, it is expected to provide useful tools to treat a wide range of diseases and disorders amenable to treatment by modifying the expression endogenous genetics. • There remains a long felt need in the art for better tools and methods for the delivery of siRNAs and other small inhibitory nucleic acids (siRNAs) into cells, particularly in view of the fact that existing techniques for the delivery of nucleic acids to cells are limited by the low efficiency and / or high toxicity of the supply reagents. There are related needs for improved methods and formulations for the supply of siRNAs in an effective amount, in an active and durable state, and using non-toxic delivery vehicles, to selected cells, tissues or compartments to mediate the regulation of the genetic expression of a way that will alter a phenotype or state of disease of the target cells. SUMMARY OF THE INVENTION One aspect of the present invention is a composition comprising a polypeptide that enhances the delivery of polynucleotides and a double-stranded ribonucleic acid (dsRNA), wherein the polypeptide that improves the supply of polynucleotides is amphipathic and comprises binding properties to nucleic acid. In a related embodiment, the polypeptide that enhances the polynucleotide delivery comprises from about 5 to about 40 amino acids, and has all or part of a sequence selected from the group consisting of Poly (Lys, Tryp) 4: 1 MW 20,000-50,000 , Poly (Orn, Trp) 4: 1 20,000-50,000, Melitin, Histone Hl, Histone H3 and Hi.stone H4, SEQ ID Nos 27 to 31, 35 to 42, 45, 47, 50 to 59, 62, 63, 67, 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178 and 180 to 186. In another embodiment, the composition causes the uptake of dsRNA into an animal cell. In another embodiment, the animal cell is a mammalian cell. In another embodiment, the composition is administered to an animal. In a related embodiment, the animal is a mammal. In another embodiment, the N-terminus of the polypeptide that improves the polynucleotide delivery is acetylated. In a related embodiment, the N-terminus of the polypeptide that improves the polynucleotide delivery is pegylated. Still in another embodiment, dsRNA is a small, interfering ribonucleic acid (siRNA) consisting of a sequence of about 10 to about 40 base pairs that is complementary to a portion of a tumor necrosis factor-alpha gene (TNF-). In a related embodiment, the dsRNA is an siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID Nos 109 to 163 and 187. In another embodiment, the polypeptide that improves delivery of polynucleotides is mixed, complexed or conjugated to the dsRNA. In another embodiment, the polypeptide that enhances the polynucleotide delivery binds to the dsRNA. In yet another embodiment, any of the above compositions further comprises a cationic lipid. In a related embodiment, the cationic lipid is selected from the group consisting of N- [1- (2,3-dioleoyloxy) propyl] chloride. -N, N, -trimethylammonium, 1,2-bis (oleoyloxy) -. 3, 3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2- (boxamido) silylamine) ethyl] -N, N-dimethyl trifluoroacetate -l-propanaminium, l, 3-dioleoyloxy-2- (6-carboxyespermil) -propylamide, 5-carboxymethylglycine dioctadecylamide, tetramethyltetrapalmitoyl spermine, tetramethyltetraoleyl spermine, tetramethyltetralauryl sperm, tetramethyltetramiristyl spermine and tetramethyldioleyl spermine, DOTMA (N- [1- (2, 3-dioleoyloxy) propyl] -N, N, N-trimethyl ammonium chloride, DOTAP (1,2-bis (oleoyloxy) -3, 3- ( trimethylammonium) propane), D RIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide), DDAB (dimethyl dioctadecyl ammonium bromide), cationic polyvalent lipids, lipoespermines, DGSPA (trifluoroacetate (2,3 -dioleyloxy-N- [2-sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium), DOSPER (1,3-dioleoyloxy-2- (6-carboxymethyl) -propyl-amide, di- and tetra-alkyl-tetra-methyl spermine, TMTPS (tet.ramétiltetrapalmitoil espermina), T TOS (tetramethyltetraoleyl spermine), TMTLS · (Tetramethyltetralauryl spermine), 'TMTMS (tetramethyltetramiristyl spermine), TMDOS (tetramethyldioleyl spermine), DOGS (dioctadecyl-amidoglycyl spermine) (TRANSFECTAM®), cationic lipids combined with non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DphPE (difitanoylphosphatidylethanolamine) or cholesterol, a cationic lipid composition composed of a 3: 1 (w / w) mixture of DOSPA and DOPE, and a 1: 1 (weight / weight) mixture of DOTMA and DOPE. In another aspect of the present invention, there is found a method for causing the absorption of a double-stranded ribonucleic acid (dsRNA) in an animal cell, which comprises incubating the animal cells with a mixture that comprises a polypeptide that enhances the delivery of polynucleotides and dsRNA, wherein the polypeptide that improves the polynucleotide delivery is amphipathic and comprises nucleic acid binding properties. In another aspect of the present invention there is found a method for modifying the expression of a target gene in an animal cell, comprising incubating the animal cell with a mixture comprising a polypeptide that enhances the delivery of polynucleotides, wherein the polypeptide which improves the polynucleotide supply is amphipathic and comprises nucleic acid binding properties, and. a double-stranded ribonucleic acid (dsRNA), where the dsRNA is complementary to a region of the target gene. In a related embodiment, in any of the above methods, the animal cell is a mammalian cell. In another aspect of the present invention, there is found a method for changing a phenotype of an animal subject, comprising administering to the animal subject a mixture of a polypeptide that enhances the polynucleotide delivery, wherein the polypeptide that improves the polynucleotide delivery is amphipathic and comprises binding properties to nucleic acid, and a double-stranded ribonucleic acid (dsRNA), where the dsRNA is complementary to a region of a target gene in the subject. In a related embodiment, the animal can be a mammal. In another embodiment, in any of the above methods, the polypeptide that enhances the polynucleotide delivery comprises from about 5 to about 40 amino acids, and has all or part of a sequence selected from the group consisting of Poly (Lys, Tryp) 4: 1 MW 20,000-50,000, Poly (Orn, Trp) 4: 1 20,000-50,000, Melitin, Histone Hl, Histone H3 and Histone H4, SEQ ID Nos 27 to 31, 35 to 42, 45, 47, 50 to 59, 62 , 63, 67, 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178 and 180 to 186. In a related embodiment, in any of the above methods, the N-terminus of the polypeptide which improves the supply of polynucleotides is acetylated. In another related embodiment, in any of the above methods, the N-terminus of the polypeptide that improves the polynucleotide delivery is pegylated. In another embodiment, in any of the above methods, dsRNA is a small interfering ribonucleic acid (siRNA) consisting of a sequence of about 10 to about 40 base pairs that is complementary to a portion of the alpha-factor gene of tumor necrosis (TNF-). In a related embodiment, in any of the above methods, the dsRNA is an siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID Nos 109 to 163 and 187. In another embodiment, in any of the above methods, the polypeptide enhancing the polynucleotide delivery is mixed, complexed or it is conjugated to the ARNds. In yet another embodiment, in any of the above methods, the polypeptide that enhances the polynucleotide delivery binds to the dsRNA. In another embodiment, in any of the above methods, further comprising a cationic lipid. In a related embodiment, in any of the above methods, the cationic lipid is selected from the group consisting of N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride, 1, 2 -bis (oleoyloxy) -3, 3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2,2-trifluoroacetate, 3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-l-propanaminium, l, 3-dioleoyloxy-2- (6-carboxyespermil) -propylamide, 5-carboxymethylglycine dioctadecylamide, tetramethyltetrapalmitoyl spermine, tetramethyltetraoleyl spermine, tetramethyltetralauryl spermine, tetramethyltetramiristyl spermine and tetramethyldioleyl spermine, DOTMA chloride of (N- [1- (2, 3-dioleoyloxy) propyl] -?,?,? - trimethyl ammonium, DOTAP (1,2-bis (oleoyloxy) -3,3- (trimethylammonium) propane), DMRIE (bromide) of 1,2- dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium), DDAB (dimethyl dioctadecyl ammonium bromide), polyvalent cationic lipids, lipoespermines, DOSPA ((2,3-dioleyloxy-N- [2-sperminecarboxamido) ethyl] -N trifluoro acetate, N-dimethyl-1-propanaminium), DOSPER (1,3-dioleoyloxy-2- (6-carboxy spermyl) -propyl-amide, di- and tetra-alkyl-tetra-methyl spermines, TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS ( tetramethyltetraoleyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMS (tetramethyltetramiristyl spermine), TMDOS (tetramethyldioleyl spermine), DOGS (dioctadecyl-amidoglycyl spermine) (TRANSFECTA ®), cationic lipids combined with non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine),. DphPE (difitanoylphosphatidylethanolamine) or cholesterol, a cationic lipid composition composed of a 3: 1 (w / w) mixture of DOSPA and DOPE, and a 1: 1 (w / w) mixture of DOTMA and DOPE .. In another aspect of the invention, is the use of a mixture comprising a polypeptide that enhances the polynucleotide delivery, wherein the polypeptide that improves the polynucleotide delivery is amphipathic and comprises binding properties to nucleic acid, and a double-stranded ribonucleic acid ( DsRNA) for the production of a medicament for the treatment of an inflammatory condition (s) associated with necrosis factor-alpha tumor (TNF-) in an animal subject, in. wherein the medicament is capable of reducing TNF-a RNA levels, thus preventing or reducing the occurrence or severity of one or more symptoms of the inflammatory condition (s) associated with TNF-OI. In one embodiment, the polypeptide that improves the polynucleotide delivery comprises from about 5 to about 40 amino acids, and has all or part of a sequence selected from the group consisting of Poly (Lys, Tryp) 4: 1 MW 20,000-50,000, Poly (Orn, Trp) 4: 1 20,000-50,000, Melitin, Histone Hl, Histone H3 and Histone H4, SEQ ID Nos 27 to "31, 35 to 42, 45, 47, 50 to 59, 62, 63, 67, 68 , 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178 and 180 to 186. In a related embodiment, the N-terminus of the polypeptide that improves the polynucleotide delivery is acetylated. , the N-terminus of the polypeptide which improves the polynucleotide delivery is pegylated In another embodiment, the dsRNA is a small interfering ribonucleic acid (siRNA) consisting of a sequence of about 10 to about 40 base pairs which is complementary to a portion of the tumor necrosis factor-alpha gene (TNF-a). Once the dsRNA is an siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID Nos 109 to 163 and 187. In another embodiment, the polypeptide that improves the Polynucleotide supply is mixed, complexed or conjugated to the dsRNA. In another embodiment, the polypeptide that enhances the polynucleotide delivery binds to the dsRNA. In another embodiment, the animal subject is a mammal. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the peptide-mediated uptake and the effect on cell viability of siRNAs complexed or conjugated to a polypeptide that enhances the polynucleotide delivery of the invention (SEQ ID NO: 35). Cellular uptake and cell viability are expressed as a percentage. Figure 2 further illustrates the peptide-mediated uptake of ARNSis complexed or conjugated to a polypeptide that improves the delivery of polynucleotides of the invention (SEQ ID NO: 35). Cellular absorption is expressed as mean fluorescent intensity (MFI). Figure 3 shows the peptide-mediated uptake of the siRNAs in human monocytes with several different polynucleotide delivery enhancement polypeptides. Figure 4 illustrates the effect on the viability of human monocytes after exposure to siRNAs complexed with several different polynucleotide delivery enhancement polypeptides. Figure 5 shows that mice injected with SiRNA / peptide have a delayed RA progress comparable to that exhibited by subjects treated with Ramicade. The progress of RA was measured by a paw evaluation index. Figure 6 provides results of absorption efficiency and viability studies in mouse tail fibroblast cells for the rationally engineered PN73 derived polynucleotide delivery enhancement polypeptides of the invention. Figure 7 illustrates that the peptide-mediated uptake of the siRNAs complexed to a polypeptide that improves the polynucleotide delivery of the invention does not emit an interferon response compared to the lipofectamine-mediated delivery of the siRNAs. (A): siRNA complexed with lipofectamine (B): siRNA complexed with PN73 (1: 5). Figure 8 shows that siRNAs conjugated to a polypeptide that improves; the polynucleotide delivery has a higher in vitro deactivation activity than siRNAs complexed with a polypeptide that improves the supply of polynucleotides. Figure 9 shows a comparison of cellular uptake between the cholesterol-conjugated siRNAs and non-conjugated siRNAs with a polypeptide that improves the supply of polynucleotides.

Figure 10 shows. that the inhibition in serum of the cellular uptake of cholesterol-conjugated siRNAs can be rescued with a polypeptide that improves the supply of polynucleotides. DESCRIPTION OF EXEMPLARY MODALITIES OF THE INVENTION The present invention satisfies these needs and fulfills the additional objects and advantages by providing new compositions and methods employing a short interfering nucleic acid (ANsi), or a precursor thereof, in combination with a polypeptide which improves the supply of polynucleotides. The polypeptide that enhances the polynucleotide delivery is a natural or artificial polypeptide · selected for its ability to improve intracellular delivery or absorption of polynucleotides, including ANsis and its precursors. Within the novel compositions of the invention, the ANsi can be mixed or complexed with, or conjugated to, the polypeptide that enhances the polynucleotide delivery to form a composition that improves the intracellular delivery of the ANsi compared to the delivery resulting from the contact target cells with a naked ANSI (ie, ANsi without the present enhancement polypeptide). In certain embodiments of the invention, the polypeptide that improves the supply of polynucleotides is a histone protein or a polypeptide or peptide fragment, derivative, analog or conjugate thereof. Within these embodiments, the ANSI is mixed, complexed form, or conjugated with one or more full length histone proteins or polypeptides corresponding at least in part to a partial sequence of a histone protein, for example one or more of the following histones: histone Hl, histone H2A, histone H2B, histone H3 or histone H4, or one or more polypeptide fragments or derivatives thereof comprising at least a partial sequence of a histone protein, typically at least 5- 10 or 10-20 contiguous residues of a native histone protein. In more detailed embodiments, the mixture, complex or conjugate of siRNA / histone is. finds substantially free of antipathetic compounds. In other detailed embodiments, the ANsi is mixed, complexed, or conjugated with the histone protein or the polypeptide will comprise a double-stranded RNA, for example 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 below. Even in additional detailed embodiments, the polypeptide that enhances the polynucleotide delivery can be found pegylated to improve stability and / or efficacy, particularly in the context of in vivo administration. Within the additional embodiments of the invention, the polypeptide that enhances the polynucleotide delivery is selected or rationally designed to comprise an amphipathic amino acid sequence. For example, polypeptides that enhance the delivery of useful polynucleotides comprising a plurality of non-polar or hydrophobic amino acid residues that form a hydrophobic sequence domain or motif linked to a plurality of charged amino acid residues that form a domain or motif can be selected. of charged sequence that produces an amphipathic peptide. In other embodiments, the polypeptide that enhances the polynucleotide delivery is selected to comprise a protein transduction domain or motif and a fusogenic peptide domain or motif. A protein transduction domain is: a peptide sequence that is capable of being inserted into and preferably transiting through the membrane of cells. A fusogenic peptide is a peptide that destabilizes a lipid membrane, for example a plasma membrane or the membrane surrounding an endosome, which can be improved at low pH. Exemplary fusogenic domains or motifs are found in a wide variety of viral fusion proteins and other proteins, for example, factor 4 of fibroblast growth (FGF4). To rationally design the polynucleotide delivery enhancement polypeptides 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 the endosomes has a low pH that results in the fusogenic peptide motif that destabilizes the membrane of an endosome. * The destabilization and disruption of an endosome membrane allows the release of the ANsi within the cytoplasm where the ANsi can associate with a PISC complex and target its target mRNA. Examples of protein transduction domains for. Optimal incorporation into the polynucleotide delivery enhancement polypeptides of the invention include: 1. TAT protein transduction domain (PTD) (SEQ ID NO: 1) KRRQRRR; 2. Penetratin PTD (SEQ ID NO: 2) RQIKI FQNRRMKWKK; 3. VP22 PTD (SEQ ID NO: 3) DAATATRGRSAASRPTERPRAPARSASRPRRPVD; 4. Kaposi sequences of FGF signal (SEQ ID NO: 4) AAVALLPAVLLALLAP, and (SEQ ID NO: 5) AAVLLPVLLPVLLAAP; 5. Integrin beta3 human signal sequence (SEQ ID NO: 6) VTVLALGALAGVGVG: 6. gp41 fusion sequence (SEQ ID NO: 7) GALFLGWLGAAGST GA; 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; 11. Arginine peptide (SEQ ID NO: 12) RRRRRRR; Y 12. Peptide of amphiphilic model (SEQ ID NO: 13) KLALKLALKALKAALKLA. Examples of fusogenic domains of viral fusion peptides for. optional incorporation into the polynucleotide delivery enhancement polypeptides of the invention, include: 1. Influenza HA2 (SEQ ID NO: 14) GLFGAIAGFIENGWEG; 2. Sendai Fl (SEQ ID NO: 15) FFGAVIGTIALGVATA; 3. Fl respiratory syncytial virus (SEQ ID NO: 16) FLGFLLGVGSAIASGV; 4. HIV gp41 (SEQ ID NO: 17) GVFVLGFLGFLATAGS; and 5. Ebola GP2 (SEQ ID NO: 18) GAAIGLA IPYFGPAA. Even within the additional modalities of the. invention, polynucleotide delivery enhancement polypeptides are provided that incorporate a DNA binding domain or motif that facilitates the formation of the polypeptide-ANSI complex and / or that enhances the supply of ANsis within the methods and compositions of the invention. Exemplary DNA binding domains in this context include various "zinc finger" domains as described for the DNA binding regulatory proteins and other proteins identified in Table 1, below (see, eg, Simpson et al., J. Biol. Chem., 278: 28011-28018, 2003).

Table 1: Zinc Finger Motifs Exemplary of Different Proteins DNA binding Zinc indicator motive .... | .... | .... J .... Í I .... | .... | . . . . I. . . . I. . . . \,.,. \ 665 G7S 685 695 705 715 ? PiP ACTGPTCKDS EGRGSG DPGKKKQBIC HIÜSCGKyYG Sp2 IIs GEQGKKKEYC HHDCGKTK LH ± GER- Sp3 GCF AGTCENCK1G GGRGTtí -Z-GKESÍQKC HIPGCGKWG KESEQ-Rahi-spi & CSCFNCHEG EGRSSbT- EPGKKK3RX0 HIEGCGWG DrosBtd RCTCENCHÍE MSGtPPIVCrp DERGSKDEIC HIFGCERLYG KJKS-EC-Siam WHi'GS PFLC Bros¾ > TCDCEHCQES. ERLGEAjGV- HLRIRIJrHSC HE? SCGXWG iccsiamHi-R WH £ GEai? FVC Cer22CB.5 K3JG DRGSCSTELC SVPGCGKTYK KFSEI-RáH3-S KH5GDRFPVC Y40B1 &.4 PQISLKKm 'FFTFSMFR- GDGSR2¾XC KTSrEL_ftAHm GaftGKKK¾C Pro-site model C- x (2,4) - C - x (12) - H - x (3) -H * The table demonstrates a conservative zinc finger motif for double-stranded DNA binding characterized by the motif pattern Cx (2, 4) -Cx (12) -Hx (3) -H (SEQ ID NO: 188) , which by itself can be used to select and design additional polynucleotide supply enhancement polypeptides according to the invention. ** To the sequences shown in Table 1, for Spl, Sp2, Sp3, Sp4, DrosBtd, DrosSp, CeT22C8.5 ,. and Y4pBlA.4, they are assigned SEQ ID Nos: 19, 20, 21, 22, 23, 24, 25 and 26, respectively. Alternatively, the DNA binding domains useful for constructing the polynucleotide delivery enhancement polypeptides 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, the polynucleotide delivery enhancement polypeptides can be designed and rationally constructed by combining any of the structural elements, domains or prior motifs, within a single effective polypeptide to mediate the improved delivery of ANsis within target cells. For example, a protein transduction domain of the TAT polypeptide was fused to the 20 N-terminal amino acids of the influenza virus hemagglutinin protein, designated HA.2, to produce a polypeptide that enhances the supply of polynucleotides in the I presented. In the present description, various other constructs of the polypeptide that enhance the polynucleotide delivery are provided, evidencing that the concepts of the invention are widely applicable to create and use a diverse array of effective polynucleotide delivery enhancement polypeptides to improve the supply of the ANsi . The polypeptides for improving the supply of Additional exemplary polynucleotides within the invention can be selected from the following peptides: WWETWKPFQCRICMRNFSTRQARRNHRRRHR (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 Poli | Orn-Trp,: 1, MW 20, 000-50, 000. Additional polynucleotide delivery enhancement polypeptides that are useful within the compositions and methods herein, comprise all or part of the protein sequence melitina. In the examples below, other exemplary polynucleotide delivery enhancement polypeptides are still identified. Any or a combination of these peptides can be selected or combined to produce polypeptide reagents that enhance the delivery of effective polynucleotides to induce o- facilitate the intracellular delivery of ANsis within the methods and compositions of the invention. In more detailed aspects of the invention, the mixture, complex or conjugate comprising an siRNA and a polypeptide that improves the supply of polynucleotides can optionally be combined with (e.g., mixed or complexed with) a cationic lipid, such as LIPOFECTIN®. In this context, it is unexpectedly described here, that the Polynucleotide delivery enhancement polypeptides complexed or conjugated to an siRNA alone will effect sufficient ANSI delivery to mediate silencing of the gene by RNAi. However, it is further unexpectedly disclosed herein that urt complex or siRNA / polypeptide conjugate that enhances polynucleotide delivery will exhibit even greater activity in mediating the delivery of ANsi and silencing of the gene upon mixing or complexing with a cysionic lipid, such as lipofectin. To produce these compositions comprised of a polypeptide that enhances the delivery of polynucleotides, siRNA and a cysionic lipid, the siRNA and the peptide can be mixed together first in a suitable medium such as a cell culture medium, after which, it is added the cysionic lipid to the mixture to form a siRNA / delivery peptide / cytiotic lipid composition. Optionally, the peptide and the cythionic lipid can be mixed between. if first in a suitable medium such as a cell culture medium, after which the siRNA can be added to form the siRNA / delivery peptide / cytiotic lipid composition. Examples of cationic lipids useful 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- bromide dimyristyloxypropyl-3-dimethylhydroxyethylammonium, and dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-l-propanaminium trifluoroacetate, 1,3-dioleoyloxy-2- (6 -carboxyespermil) -propylamide, 5-carboxymethylglycine dioctadecylamide, tetramethyltetrapalmitoyl spermine, tetramethyltetradecyl spermine, tetramethyltetralauryl spermine, tetramethyltetramiristyl spermine and tetramethyldioleyl spermine, DOTMA (N- [1- (2, 3-dioleoyloxy) propyl] -N, N chloride, N-trimethyl ammonium, DOTAP (1, 2-bis (oleoyloxy) -3, 3- (trimethylammonium) propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide), or DDAB (bromide of dimethyl dioctadecyl ammonium.) Cationic polyvalent lipids include, lipoespermines, DOSPA (2,3-dioleyloxy-N- [2-sperminecarboxamido) ethyl] -N, N-dimethyl-l-propanaminium trifluoroacetate), and DOSPER (1 , 3-dioleoyloxy-2- (6-carboxy-spermyl) -propyl-amide, di- and tetra-alkyls uil-tetra-methyl spermines, including but not limited to TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMS (tetramethytetramiristyl spermine), and TMDOS (tetramethyldioleyl spermine), DOGS (dioctadecyl-amidoglycyl spermine) (TRANSFECTAM®) Other useful cationic lipids are described, for example, in U.S. Patent No. 6,733,777; Patent of E.U. No. 6,376,248; Patent of E.U. No. 5,736,392; Patent of E.U. No. 5,686,958; Patent of E.U. 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 DOPE (dioleoylphosphatidylethanolamine), DphPE (difitanoylphosphatidylethanolamine) p cholesterol. A cationic lipid composition composed of a 3: 1 (w / w) mixture of DOSPA and DOPE or a 1: 1 (w / w) mixture of DOTMA and DOPE (LIPOFECTIN®, Invitrogen) is generally useful for transfecting the compositions of this invention. Preferred transfection compositions are those that induce a substantial transfection of a larger eukaryotic cell line. In exemplary embodiments, the present invention features compositions comprising a small nucleic acid molecule, such as short interfering nucleic acid (ANsi), a short interfering RNA (siRNA), a double-stranded RNA (dsRNA), micro-RNA ( MRNA) or a short capillary RNA (shRNA), mixed or complexed with, or conjugated to, a polypeptide that enhances the supply of polynucleotides. As used herein, the term "short interfering nucleic acid", "ANsi", "short RNA of "interference", "siRNA", "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule" or "chemically modified short interfering nucleic acid molecule", refers to a nucleic acid molecule capable of inhibiting or sub-regulating gene expression or viral replication, for example, by interfering with "RNAi" RNA or silencing the gene in a sequence-specific manner. Within exemplary embodiments, the ANsi is a double-stranded polynucleotide molecule that comprises self-complementary regions of sense and antisense, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule for sub-regulating expression, or a portion thereof , and the sense region comprises a sequence of nucleotides corresponding to (ie, which is substantially identical in sequence). n) a) the target sequence of nucleic acid, or a portion: thereof. "ANsi" means a small interfering nucleic acid, for example an siRNA, ie, it is a short-stranded nucleic acid of short length (or optionally a larger precursor thereof), and that it is not unacceptably toxic in target cells. The length of useful ANsis within the invention will be optimized, in certain modalities, to a length of approximately 21 to 23 bp in length. However, there is no particular limitation on the length of ANsis. useful, including siRNA. For example, ANsys may initially present to the cells in a precursor form that is substantially different from the final or processed form of the ANsi that will exist and exert the silencing activity of the gene when delivered, or after being delivered, to the targeted cell. . The precursor forms of ANsis, for example, may include precursor sequence elements that are processed, degraded, altered or split at or after the delivery time to produce an ANsi that is active within the cell to mediate the silencing of the gen. Thus, in certain embodiments, useful ANsys 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 that will produce an ANsi active, processed, within the target cell. In other embodiments, an ANsi or useful ANsi precursor will be from about 10 to 49 bp, from 15 to 35 bp or from about 21 to about 30 bp in length. In certain embodiments of the invention, as noted above, polynucleotide delivery enhancement polypeptides are used to facilitate delivery of larger nucleic acid molecules than Conventional ANsis, including large nucleic acid precursors of ANsis. For example, the methods and compositions herein may be employed to enhance the delivery of larger nucleic acids that represent "precursors" to the desired ANs, where the precursor amino acids may be cleaved or otherwise processed before, during or after delivery to a target cell to form an active ANsi to modulate the genetic expression within the target cell. For example, an ANsi precursor polynucleotide can be selected as a single-stranded circular polynucleotide having two or more loop structures and one or more roots 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 a sequence of nucleotides corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active ANsi molecule capable of mediating RA i. In mammalian cells, dsRNAs longer than 30 base pairs can activate PKR of dsRNA-dependent kinase and 2'-5'-oligoadenylate synthetase, normally induced by interferon. Activated PKR inhibits thegeneral translation by the phosphorylation of the factor 2a of eukaryotic initiation of the translational factor (eIF2a), while the 2'-5'-oligoadenylate synthetase. causes nonspecific degradation of mRNA by activation of RNase L. by virtue of its small size (with particular reference to non-precursor forms), commonly, less than 30 base pairs and more commonly between about 17-19, 19 -21 or 21-23 base pairs, the ANsis of the present invention prevent the activation of the response to interferon. In contrast to the nonspecific effect of the long dsRNA, the siRNA can mediate the selective silencing of the gene in the mammalian system. Capillary RNAs, with a short loop and from 19 to 27 base pairs in the root, also selectively silence the expression of genes that are homologous to the sequence in the double-stranded root. Mammalian cells can convert short hair RNA into an siRNA to mediate selective silencing of the gene. RISC mediates the unfolding of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. The cleavage of the target RNA takes place in the middle part of the region complementary to the antisense chain of the siRNA dupl. Studies have shown that 21-nucleotide siRNA pairs are more active when they contain two overhangs 3 'of nucleotide. In addition, the complete substitution of one or both strands of siRNA with 2'-deoxy (2'H) or 2'-O-methyl nucleotides. it avoids the activity of RNAi, whereas it has been reported that the replacement of the nucleotides with protrusion of the 3 'terminal siRNA with nucleotides deoxy (2'-H) is tolerated. Studies have shown that replacement of the 3 'overhang segments of a 21 mer siRNA pair that have 2 nucleotide 3' overhangs with deoxyribonucleotides does not have an adverse effect on the activity of AR i. It has been reported that replacement of up to 4 nucleotides at each end of the siRNA with deoxyribonucleotides is highly tolerated whereas complete substitution with deoxyribonucleotides results in no RNAi activity. Alternatively, the siRNAs can be delivered as single or multiple transcription products expressed by a polynucleotide vector encoding single or multiple ANs and directing their expression within the target cells. In these embodiments, the double-stranded portion of a final transcription product of the siRNAs to be expressed within the target cell, for example, can be 15 to 49 bp, 15 to 35 bp, or approximately 21 to 30 bp of length. Within the exemplary modalities, the double-stranded portions of the ANsis, in which two chains are paired, are not limited to completely matched nucleotide segments, and may contain unpaired portions due to incompatibility, (the corresponding nucleotides are not complementary), bent (lack of the corresponding complementary nucleotide in a chain ), outgoing and similar. The unpaired portions may be contained to the extent that they do not interfere with the formation of ANsi. In more detailed embodiments, a "bent" may comprise 1 to 2 unpaired nucleotides, and the double stranded region of the ANsis in which two chains are paired may contain from about 1 to 7, or from about 1 to 5 bent. · Additionally, the "incompatible" portions contained in the double-stranded region of the ANsis may be present in numbers from about 1 to 7, or from about 1 to 5. More frequently, in the case of incompatibilities, one of the nucleotides is guanine, and the other is uracil. Such incompatibility can be attributed, for example, to a mutation from C to T, from G to A, or mixtures thereof, into a corresponding DNA encoding sense RNA, but other causes are also contemplated. Furthermore, in the present invention, the double-stranded region of the ANsis in which two chains are paired, may contain both bent and incompatible portions in the numerical ranges approximate specified. The terminal structure of the ANsis of the invention can be either blunt or. cohesive (outgoing) while the ANsi retains its activity to silence the expression of target genes. The cohesive (protruding) end structure is not limited to the 3 'overhang only as reported by others. In contrast, the 5 'overhang structure can be included as long as it is able to induce a silencing effect of the gene such as by RNAi. Additionally, the number of salient nucleotides is not limited to the reported limits of 2 or 3 nucleotides, but can be any number provided that the salient does not impair the silencing activity of the ANsi gene. For example, the projections may comprise from about 1 to 8 nucleotides, more frequently from about 2 to 4 nucleotides. The total length of ANsis having a cohesive end structure is expressed as the sum of the length of the paired double-stranded portion and that of a pair comprising single strands projecting at both ends. For example, in the exemplary case of a 19 bp double-stranded RNA with 4 nucleotide overhangs on both sides, the total length is expressed as 23 bp. In addition, since the outgoing sequence may have low specificity for a target gene, it is not necessarily complementary (antisense) or identical (sense) to the sequence of the target gene. Also, always that the ANsi be able to maintain its effect of silencing the gene in the target gene, may contain a low molecular weight structure (eg, a natural RNA molecule such as tRNA, rRNA or viral RNA, or the artificial RNA molecule) ), for example, in the outgoing portion at one end. Additionally, the terminal structure of the ANsis may have a root-loop structure in which the ends of one side of the double-stranded nucleic acid are connected by a binding nucleic acid, e.g., a binding RNA. The length of the double-stranded region (root-loop portion) can be, for example, from 15 to 49 bp, frequently from 15 to 35 bp, and most commonly from about 21 to 30 bp in length. Alternatively, the length of the double-stranded region that. is a final transcription product of A.Nsis to be expressed in a target cell, can be, for example, from about 15 to 49 bp, from 15 to 35 bp or from about 21 to 30 bp in length. When link segments are used, there is no particular limitation on the length of the link as long as it does not obstruct the pairing of the root portion. For example, for the stable pairing of the root portion and the suppression of recombination between the DNAs encoding this portion, the binding portion may have a trefoil leaf tRNA structure. Even if the link has a length that would obstruct the pairing of the root portion, it is possible,. For example, build the link portion to include introns so that. the introns are extracted during the processing of a precursor RNA in the mature RNA, thus allowing the pairing of the root portion. In the case of a root-loop siRNA, any end of the RNA (head or end) without loop structure, may have a low molecular weight RNA. As described above, these low molecular weight RNAs can include a natural RNA molecule, such as tRNA, rRNA or viral RNA or an artificial RNA molecule. The ANsi may also comprise a single-stranded polynucleotide having a nucleotide sequence complementary to the nucleotide sequence in a target nucleic acid molecule or a portion thereof (eg, when the ANsi molecule does not require presence within the molecule of ANsi of the nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single-stranded polynucleotide may further comprise a terminal phosphate group, such as a 5'-phosphate (see, for example, Martinez et al., Cell (Cell), 110: 563-574, 2002, and Schwarz, et al., Molecular Cell (Molecular Cell) 10: 537-568, 2002) or 5 ', 3'-diphosphate. As used herein, the term "molecule" of ANsi is not limited to molecules that contain only RNA or DNA of natural origin, but also covers chemically modified and non-nucleotide nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack nucleotides containing 2'-hydroxy (2'-OH). In certain embodiments, the short interfering nucleic acids do not require the presence of nucleotides having a 2'-hydroxy group to mediate the RNAi and therefore, the short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotide (eg. , nucleotides having a 2'-OH group). Such ANsi molecules that do not require the presence of ribonucleotides within the ANsi molecule to support the RNAi, may, however, have a bond or linked bonds and other groups, residues or linked or associated chains containing one or more nucleotides with 2'-OH groups. Optionally, the ANsi molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40 or 50% of the nucleotide positions. As used herein, the term ANsi means that it is 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), short capillary RNA (shRNA), oligonucleotide short interfering, short interfering nucleic acid, modified short interference oligonucleotide, chemically modified siRNA, post-transcriptional RNA silencing of the gene (mRNAs) and others. In other embodiments, the ANsi molecules for use within the invention may comprise separate sequences and regions of sense and antisense, wherein the sense and antisense regions are covalently linked by means of nucleotide linkage molecules or not. nucleotide, or are alternatively linked non-covalently by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and / or stacking interactions. "Antisense RNA" is an RNA strand that has a sequence complementary to an mRNA of the target gene, and is thought to induce RNAi by binding to the mRNA of the target gel. The "sense RNA" has a sequence complementary to the antisense ARN, and hybridized to its complementary antisense RNA to form the siRNA. These antisense and sense RNAs have been conventionally synthesized 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), hair RNAs, and other species of RNA that can unfold in vivo to form. ARNsis. RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of yielding transcripts that form dsRNA or capillary RNAs in cells and / or transcripts that can produce siRNAs in vivo. Optionally, the siRNA includes single strands or double strands of siRNA. A hybrid molecule, yes, is a double-stranded nucleic acid that has a similar function to siRNA. Instead of a double-stranded RNA molecule, a hybrid is comprised of an RNA strand and an AD strand. Preferably, the RNA strand is the antisense strand which is the strand that binds to the target mRNA. The hybrid if created by means of the hybridization of the DNA and RNA strands has a hybridized complementary portion and preferably at least one 3 'overhang end. ANsis for use within the invention can be assembled from two separate oligonucleotides, wherein one strand is the sense strand, and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (ie, each chain comprises a sequence of nucleotides that is complementary to the nucleotide sequence in the other chain, such as when the antisense chain and the sense chain form a duplex or double-stranded structure, for example, wherein the double-stranded region is approximately 19 base pairs). The antisense chain may comprise a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense chain may comprise a nucleotide sequence corresponding to the target acid sequence nucleic or a portion of it. Alternatively, the ANsi can be assembled from a single oligonucleotide, wherein the sense and antisense complementary regions of the ANsi are linked by means of a nucleic acid-based or non-nucleic acid-based link (s). . Within the additional modalities, the Ansis for intracellular delivery according to the methods and compositions of the invention, can be a polynucleotide with double secondary, asymmetric double, capillary or asymmetric capillary structure, having self-complementary regions of sense and antisense , wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a separate nucleic acid target molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the sequence of target nucleic acid or a portion thereof. Non-limiting examples of chemical modifications that can be produced in an ANsi include, without limitation phosphorothioate internucleotide bonds, 2'-deoxyribonucleotides, 2'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, nucleotides of " acrylic ", 5-C-methyl nucleotides, and the incorporation of abasic terminal glyceryl terminal and / or inverted deoxy. These chemical modifications, when used in various ANsi constructs, show the preservation of RNAi activity in cells while at the same time, they dramatically increase 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 the potential limitations of in vivo stability and bioavailability inherent in native RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules may allow a lower dose of a particular nucleic acid molecule for a given therapeutic effect, because the chemically modified nucleic acid molecules tend to have a longer half-life in serum. long In addition, certain chemical modifications can improve the bioavailability of the nucleic acid molecules by targeting particular cells or tissues and / or by improving the cellular uptake of the nucleic acid molecule. Accordingly, even if the activity of a chemically modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to a total RNA nucleic acid molecule, the overall activity of the acid molecule Modified nucleic acid may be greater than that of the native molecule due to the improved stability and / or delivery of the molecule. Unlike the modified native ANsi, the chemically modified ANsi can also minimize the possibility of activating interferon activity in humans. In the ANsi molecules described herein, the antisense region of an ANsi-molecule of the invention. may comprise a phosphorothioate internucleotide linkage at the 3 'end of said antisense region. In any of the embodiments of the ANsi molecules described herein, the antisense region may comprise from about one "to about five phosphorothioate internucleotide linkages at the 5 'end of said antisense region. the ANsi molecules described herein, the 3'-terminal nucleotide overhangs of an ANsi molecule of the invention may comprise ribonucleotides or deoxyribonucleotides that are chemically found modified in a sugar,. nucleic acid base or structure. In any of the embodiments of the ANsi molecules described herein, the 3 'terminal nucleotide overhangs may comprise one or more universally-based ribonucleotides. In any of the embodiments of the ANsi molecules described herein, the 3 'terminus nucleotide overhangs may comprise one or more nucleotides of acrylic. For example, in a non-limiting example, the invention features a short chemically modified interference nucleic acid (ANsi) which has approximately 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide bonds in a chain of ANsi. In yet another embodiment, the invention features a short chemically modified interference nucleic acid (ANsi). Which individually has about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both strands of ANsi. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide chains of the ANsi pair, for example, in the sense chain, in the sense chain, or in both chains. The siRNA molecules of the invention may comprise one or more phosphorothioate internucleotide linkages at the 3 'end, the 5' end, or at both 3 'and 5' ends of the sense chain, the antisense or both chains. For example, an exemplary ANSI molecule of the invention may comprise from about 1 to about 5 or more (eg, about, 1, 2, 3, 4, 5 or more) consecutive phosphorothioate internucleotide linkages at the 5 'end of the sense chain, the antisense chain, or both chains. In another non-limiting example, an exemplary ANSI molecule of the invention may comprise one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) internucleotide linkages of pyrimidine phosphorothioate in the sense chain, in the antisense chain or in both chains. In yet another non-limiting example, an exemplary ANSI molecule of the invention may comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) linkers. purine phosphorothioate internucleotide in the sense chain, in the. antisense chain or both chains. An ANsi molecule may be comprised of a circular nucleic acid molecule, wherein the ANsi is from about 38 to about 70 (eg, about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length which has about 18 to about 23 (eg, about 18, 19, 20, 21, 22 or 23) base pairs wherein the circular oligonucleotide forms a fungiform shaped structure having approximately 19 base pairs and 2 loops. A molecule of circular ANsi contains two loop motifs, wherein one or both loop portions of the ANsi molecule is biodegradable. For example, a circular ANsi molecule of the invention is designed such that degradation of the loop portions of the ANsi molecule in vivo can generate a double stranded ANsi molecule with 3 'terminal overhangs, such as protrusions. of 3 'terminal nucleotide comprising approximately 2 nucleotides. The modified nucleotides present in the ANsi molecules, preferably in the antisense chain of the 'ANsi molecules, but also optionally in the sense and / or antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to ribonucleotides of natural origin. For example, the invention features ANsi molecules that include modified nucleotides that have a Northern conformation (eg, pseudorotation Northern cycle, see, for example, Saenger, Principles of Nucleic Acid "Structure, Springer -Verlag ed., 1984) As such, the chemically modified nucleotides present in the ANsi molecules of the invention, preferably in the antisense chain of the ANSI molecules, but also optionally in sense and / or antisense and sense chains, are resistant to nuclease degradation while, at the same time, maintaining the ability to mediate AR i. Non-limiting examples of nucleotides having a Northern configuration include closed nucleic acid nucleotides (LNA) (eg, 2'-0, '-C-methylene- (D-ribofuranosyl) nucleotides;' 2 '-methoxyethoxy nucleotides (MOE) ), 2'-methyl-thio-ethyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides, and 2'-0 nucleotides The sense chain of a double-stranded ANSI molecule may have a terminal cap residue such as an inverted deoxibase residue, at the 3 'end, the 5' end or both at the 3 'and 5' ends of the Sequent chain Non-limiting examples of conjugates include the conjugates and ligands described in Vargeese et al., EU Application Serial No. 10 / 427,160, filed on April 30, 2003, incorporated by reference herein in its entirety, including In another embodiment, the conjugate is covalently bound to the molecule chemically modified ANsi using a biodegradable linker. In one embodiment, the conjugate molecule is attached to the 3 'end of the chain of sense, of the antisense chain or both chains of the chemically modified ANsi molecule. In another embodiment, the conjugate molecule is attached to the 5 'end of the sense strand, the antisense strand or both strands of the chemically modified siNA molecule. In yet another embodiment, the conjugate molecule is attached to both the 3 'end and the extreme 50 of either the sense chain, the antisense strand or both strands of the chemically modified siNA molecule, or any combination of the same. In one embodiment, the conjugate molecule of the invention comprises a molecule that facilitates the delivery of a chemically modified ANsi molecule within a biological system, such as a cell. In another embodiment, the conjugate molecule bound to the chemically modified ANsi molecule is a poly ethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the present invention that can be attached to chemically modified ANSI molecules are described in Vargeese et al., U.S. Patent Application Publication. No. 20030130186, published July 10, 2003, and the U.S. Patent Application Publication. No. 20040110296, published on June 10, 2004. The type of conjugates used and the amount of conjugation of the molecules of ANSI of the invention can be evaluated by improved pharmacokinetic profiles, bioavailability and / or stability of ANsi constructs, while, at the same time, maintaining the ability of the ANsi to mediate the activity of RNAi. As such, those skilled in the art can visualize ANsi constructs that are modified with several conjugates to determine whether the ANsi conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as is generally known in the technique. In addition, an ANsi can be understood in addition to a nucleotide linker, non-nucleotide linker or mixed nucleotide / non-nucleotide linker that binds the sense region of the ANsi a., The antisense region of the ANsi. . In one embodiment, the nucleotide linker can be a " linker " 2 nucleotides in length, for example, of 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 target molecule in its natural disposition. Alternatively, an aptamer can be an acid moleculenucleic that binds to a target molecule in which the target molecule does not naturally bind to a nucleic acid. The molecule, target 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 the interaction of the naturally occurring ligand with the protein. this is a non-limiting example and the technicians 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; Kusser, J, Biotechnol., 74:27, 2000; Hermann and Patel, Science 287: 820, 2000; and Jayasena, Clinical Chemistry 45: 1628, 1999. A non-nucleotide linkage can comprise an abasic nucleotide, polyether, polyamide, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (eg, polyethylene glycols such as those having between 1 and 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., Biochemist.ry 30: 9914, 1991; Arnold et al., International Publication No. WO 89/02439; Usman 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" also means any group or compound that can be incorporated into a chain of nucleic acid in the place of one or more nucleotide units, including substitutions of either sugar and / or phosphate, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymidine, for example in the Cl position of the sugar. The synthesis of an ANsi molecule of the invention, which can be chemically modified, comprises: (a) the synthesis of two complementary strands of the ANsi molecule; (b) hybridization of the two complementary strands together under suitable conditions to obtain a double-stranded ANSI molecule. In another embodiment, the synthesis of the two complementary strands of the ANsi molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, the synthesis of the two complementary strands of the ANsi molecule is by oligonucleotide synthesis in solid phase series. Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., Methods in Enzymology 211: 3-19, 1992; Thompson et al., International PCT Publication No. WO 99/54459; incott et al., Nucleic Acids Res., 23: 2677-2684, 1995; Wincott et al., Methods Mol. Bio. 74:59, 1997; Brennan et al., Biotechnol Bioeng., 61: 33-45, 1998; and Brennan Patent of E.U. No. 6,001,311. RNA synthesis, including certain ANsi molecules of the invention, follows the general procedures described, for example, in Usman et al., J. Am. Chem. Soc, 109: 7845, 1987; Scaringe et al., Nucleic. Acids Res., 18: 5433, 1990; and Wincott et al., J. Am. Chem. Soc, 109: 7845, 1987; Scaringe et al., Nucleic Acids Res., 18: 5433, 1990; and Wincott et al., Nucleic Acids Res., 23: 2677-2684, 1995; Wincott et al., Methods Mol .. Bio., 74:59, 1997. Supplementary or complementary methods for the delivery of 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 (Delivery Strategies for Therapeutic Oligonucleotide antisense), 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. Sullivan et al., International PCT Publication No. WO 94/02595, further discloses general methods for the delivery of enzymatic nucleic acid molecules. These protocols can be used to supplement or complement the delivery of virtually any nucleic acid molecule contemplated within the invention. Nucleic acid molecules and polynucleotide delivery enhancement polypeptides can be administered to cells by a variety of methods known to those skilled in the art, including, but not limited to, administration within formulations comprising the ANsi and the polypeptide which enhances the supply of polynucleotides alone, or which further comprise one or more, additional components such as a carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, and the like pharmaceutically acceptable. In certain embodiments, the ANsi and / or the polypeptide that improves the polynucleotide delivery can be encapsulated in liposomes, administered by iontophoresis, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (see, e.g., 0 'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, a combination of nucleic acid / peptide / vehicle can be delivered locally by direct injection or by the use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., 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 effectively employed as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence or severity of, or treat (alleviate one or more of the symptoms to a detectable or calculable extent) a disease state or other adverse condition in a patient. Therefore, within the additional embodiments, the invention provides pharmaceutical compositions and methods that characterize the presence or administration of one or more polynucleic acids, typically one or more ANsys, combined, complexed or conjugated with a polypeptide that improves delivery of polynucleotides, optionally formulated with a pharmaceutically acceptable carrier such as a diluent, stabilizer, buffer and the like. The present invention satisfies the additional objects and advantages by providing short nucleic acid interference (ANSI) molecules that modulate the expression of genes associated with a particular disease state or other adverse condition in a subject. Typically, ANSI will direct a gene that is expressed at a high level as a causal factor or contributor associated with the disease state or adverse condition of the subject. In this context, ANSI will effectively sub-regulate gene expression at levels that prevent, alleviate or reduce the severity or recurrence of one or more associated disease symptoms. Alternatively, for various different disease models where the target gene expression is not necessarily elevated as a consequence or sequelae of the disease or other adverse condition, the sub-regulation of the target gene will result, however, in a therapeutic result decreasing the genetic expression (ie, to reduce the levels of a selected product of mRNA and / or protein of the target gene). Alternatively, the ANsis of the invention can be targeted to decrease the expression of a gene, which can result in up-regulation of a "downstream" gene whose expression is negatively regulated by of a product or activity of the target gene. Within the exemplary embodiments, the compositions and methods of the invention are useful. as therapeutic tools to regulate the expression of tumor necrosis factor-alpha TNF-OI 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 a TNF-OI by RNA interference (RNAi) using small nucleic acid molecules. In more detailed embodiments, the invention provides small nucleic acid molecules, such as short nucleic acid-interfering molecules (ANsi), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA) ) and short hair RNA (sshRNA), and related methods that are effective to modulate the expression of a TNF-OI and / or TNF-a genes to prevent or alleviate the symptoms of RA in mammalian subjects. Within these and therapeutic compositions and related methods, the use of chemically modified ANs will frequently improve the properties of modified ANs in comparison to the properties of native ANSI molecules, for example, by providing increased resistance to nuclease degradation in vivo, and / or through improved cellular absorption. As can be easily determined in accordance with In the present description, useful Ansis having multiple chemical modifications will retain their RNAi activity. The ANsi molecules of the present invention thus provide reagents and methods useful for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering and pharmacogenomics applications. These ANss of the present invention can be administered in any form, for example, transdermally or by local injection (eg, local injection into psoriatic plaque sites to treat psoriasis, or within the joints of patients affected with psoriatic arthritis or RA). ). In more detailed embodiments, the invention provides formulations and. methods for administering therapeutically effective amounts of ANsis directed against a TNF-α mRNA, which effectively sub-regulate the TNF-α RNA and thereby reduce or prevent one or more inflammatory conditions associated with TNF-α. Comparable methods and compositions are provided that direct the 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 as aberrantly increased as a causal or contributing factor associated with the selected disease condition.

Mixtures of ANsi / polypeptide that improves the polynucleotide delivery of the invention can be administered in conjunction with other standard treatments for an objective disease condition, for example in conjunction with effective therapeutic agents against inflammatory diseases, such as RA or psoriasis. Examples of combinatorially useful and effective agents in this context include non-spheroidal anti-inflammatory drugs (NSAIDs), methotrexate, gold compounds, D-penicillamine, antimalarials, sulfasalazine, glucocorticoids and other TNF-neutralizing agents such as infliximab and Entrance The negatively charged polynucleotides of the invention (e.g., RNA or DNA) can be administered to a patient by standard means, with or. without stabilizers, buffers and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for the formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration and other compositions known in the art. The present invention also includes formulations pharmaceutically acceptable of the compositions described herein. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric acid, hydrobromic acetic acid, and benzene sulfonic acid. A "composition" or "pharmacological formulation" refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, within a cell or patient, including, for example, a human. The appropriate forms, in part, depend on the use or the route of entry, for example, oral, transdermal or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell for which delivery of the negatively charged nucleic acid is desirable). For example, pharmacological compositions injected into the blood stream must be soluble. . Other factors are known in the art, and include considerations such as toxicity. By "systemic administration" is meant the in vivo systemic absorption or accumulation of drugs in the bloodstream followed by distribution throughout the body. Routes of administration that lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, by inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the negatively charged desired polymers,. e.g., nucleic acids, to a diseased, accessible tissue. The input ratio of a drug in 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 the association of the drug with the surface of cells, such as lymphocytes and macrophages, is also useful. This method can provide an improved delivery of the drug to the target cells by taking advantage of the macrophage's specificity and immune recognition of the lymphocyte of abnormal cells, such as cancer cells. By "pharmaceutically acceptable formulation". is meant a composition or formulation that allows the effective distribution of the nucleic acid molecules of the present invention at the most appropriate 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 into the CNS (Jolliet-Riant and Tillement, Fundam Clin Pharmacol., 13: 16-26, 1999); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, DF, et al., Cell Transplant 8: 47-58 1999) (Alkermes, Inc., Cámbridge , Mass.); and charged nanoparticles, such as those produced from polybutyl cyanoacrylate, which can deliver drugs through the blood brain barrier and can alter the mechanisms of neuronal absorption (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 the material described in Boado et al., J. Pharm. Sci., 87: 1308-1315, 1998; Tyler 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; Aldrian-Herrada et al., Nucleic Acids Res. 26: 4910-4916; and Tyler et al., PNAS USA 96: 7053-7058, 1999. The present invention also includes compositions prepared for storage or administration, including a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. The vehicles or diluents acceptable for therapeutic use are well known in the pharmaceutical art and are describe, for example,. in Remington's Pharmaceutical Sciences, Mack Publishing Co., A.R. Gennaro ed., 1985. For example, condoms, stabilizers, colorants and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Additionally, antioxidants and suspending agents can be used. A pharmaceutically effective dose is that 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 experts will recognize n the medical technique. Generally, an amount between 0.1 mg / kg and 100 mg / kg of body weight / day of the active ingredients is administered depending 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, alginate. sodium, polyvinyl pyrrolidone, gum tragacanth, and acacia gum; the dispersing agents or humectants may be a phosphatide of natural origin, 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 heptadecaethylene oxyketanol, 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 anhydrides of hexitol, for example polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example,. p-hydroxybenzoate- ethyl or n-propyl, 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. The aqueous suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents may be added to provide palatable oral preparations. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing agents or humectants or suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring and coloring agents may also be present. The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil or a mineral oil or mixtures thereof. Suitable emulsifying agents can be gums of natural origin, for example, acacia gum or tragacanth gum, phosphatidás of natural origin, for example, soybeans, lecithin, and esters or partial 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. The emulsions may also contain sweetening and flavoring agents.

The pharmaceutical compositions can be in the form of a sterile aqueous or oily injectable suspension. This suspension can be formulated according to the prior art using those dispersing agents or suitable humectants and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the vehicles and acceptable solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. Additionally . Conventional sterile oils are conventionally employed as a solvent or suspending medium. For this purpose, any fixed soft oil including synthetic mono or diglycerides can be employed. Additionally, fatty acids such as oleic acid find their use in the preparation of injectables. ANsis may also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will consequently melt in the rectum to release the drug. Such materials include cocoa butter and glycols of polyethylene. ANsis can be extensively modified to improve stability by modification with nuclease-resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H. For a review, see Usman and Cedergren, TIBS 17:34, 1992; Usman et al., Nucleic Acids Symp. Ser., 31: 163, 1994. The ANsi constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography and re-suspended in water. Nucleic acid molecules chemically synthesized with modifications (base, sugar and / or phosphate) can prevent their degradation by serum ribonucleases that can increase their potency. See, e.g., Eckstein et al., International Publication No. WO. 92/07065; Perrault et al., Nature 344: 565, 1990; Pieken et al., Science, 253: 314, 1991; Usman and Cedergren, Trends in Biochem. Sci., 17: 334, 1992; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Patent No. 5,334,711; Gold et al., U.S. Patent No. 6,300,074. chemicals that can be made to the base, phosphate and / or sugar residues of the nucleic acid molecules described herein are several examples in the art that describe modifications of sugar, base and phosphate that can be introduced into the nucleic acid molecules with significant improvement in their stability and efficacy of nuclease. For example, oligonucleotides are modified to improve stability and / or improve biological activity by modification with nuclease-resistant groups, eg, base modifications of 2'-amino, 2'-C-allyl, 2 'nucleotides. -fluofo, 2'-0-methyl, 2'-H. For a review, see Usman and Cedergren, TIBS 17:34, 1992; Usman et al., Nucleic Acids Symp. Ser., 31: 163, 1994; Burgin et al., Biochemistry 35: 14090, 1996. Sugar modification of nucleic acid molecules have been extensively described in the art. See, · Eckstein, et al., Publication PCT International No. WO 92/07065; Perrault et al., Nature 344: 565-568, 1990; Pieken et al. , Science, 253: 314-317, 1991; Usman and Cedergren, Trends in. Biochem Sci. , 17: 334-339, 1992; Usman et al., PCT International Publication No. WO 93/15187; Sproat, Patent of E.U. No. 5,334,711; and Beigelman et al., J. biol. Chem., 270: 25702, 1995; Beigelman et al., PCT International Publication No. WO 97/26270; Beigelman et al., Patent of E.U. No. 5, 716, 824; Usman et al., Patent of E.U. No. 5,627,053; Woolf et al., PCT International Publication No. WO 98/13526; Thompson et al., Karpeisky et al., Tetrahedron Lett., 39: 1131, 1998; Earnsha 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 ANsi nucleic acid molecules of the present invention provided that the ability of ANsi to promote RNAi in cells is not significantly inhibited. Although the chemical modification of internucleotide bonds of oligonucleotides with phosphorothioate, phosphorodithioate and / or 5'-methylphosphonate bonds improves stability, excessive modifications may cause some toxicity and decrease in activity. Accordingly, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these bonds should reduce the toxicity, resulting in an increase in the efficiency and greater specificity of these molecules. In one embodiment, the invention features modified ANSI molecules, with modifications of phosphate structure comprising one or more substitutions. of phosphorothioate, 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" (Nucleic Acid Analogies: Synthesis and Properties in Modern Synthetic Methods), VCH, 1995, pp 331- 417, and Esmaeker et al., "Novel Backbone Replacements for Oligonucleotides in Carbohydrate Modifications' in Antisense Research" (New structure replacements for oligonucleotides in carbohydrate modifications in antisense research), ACS 1994, pp 24-39. 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 (supply strategies for 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., U.S. Patent. No. 6,395,713 and Sullivah et al., PCT O 94/02595 further describe the general methods for the delivery of nucleic acid molecules. These protocols can used for the supply of. virtually any nucleic acid molecule. The nucleic acid molecules can be administered to the cells by a. variety of methods known to those skilled in the art, including, but not limited to, encapsulation in liposomes, by iontophoresis or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see, for example, Gonzalez et al. , Bioconjugate, Chem., 10: 1068-1074, 1999, Wang et al., PCT International Publications Nos. O 03/47518 and WO 03/46185), poly (co-glycolic lactic acid) (PLGA) and microspheres of PLCA (see, for example, U.S. Patent No. 6,447,796 and U.S. Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O 'Hare and Normand, PCT International Publication No. WO 00/53722). Alternatively, the nucleic acid / vehicle combination is delivered locally by direct injection or by the use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., Clin. . Cancer Res. 5: 2330-2337, 1999, and Barry et al., PCT International Publication No. WO 99/31262. The molecules of the present invention can be used as pharmaceutical agents. The pharmaceutical agents avoid, modulate the occurrence or treat (alleviate a symptom to some degree, preferably all symptoms) of a disease state in a subject. The term "ligand" refers to any compound or molecule, such as a drug, peptide, hormone or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly. The receptor that interacts with a ligand may be present on the surface of a cell or alternatively it may be an intracellular receptor. The interaction of the ligand with the receptor can result in a biochemical reaction, or it can simply be a physical interaction or association. By "asymmetric capillary" as used herein, is meant a linear ANSI molecule comprising an antisense region, a loop portion which may comprise nucleotides or non-nucleotides and a sense region comprising fewer nucleotides than the region of antisense to the extent that the sense region has sufficient complementary nucleotides to form base pairs with the antisense region and form a loop pair. For example, the asymmetric capillary ANSI molecule of the invention may comprise an antisense region which is of sufficient length to mediate the RNAi in a T cell (eg, from about 19 to about 22 (eg, about 19, 20, 21 or 22) nucleotides and a loop region comprising from about 4 to about 8 (eg. , about 4, 5, 6, 7 or 8) nucleotides, and a sense region having from about 3 to about 18 (eg, 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 complementary capillary ANSI molecule can also comprise a 5 'terminal phosphate group which may be chemically modified. loop of the asymmetric capillary ANSI molecule may comprise nucleotides, non-nucleotides, binding molecules or conjugated molecules as described herein.As "asymmetric pair" as used herein, is meant an ANsi molecule having two chains separated that c they comprise a region of sense and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has sufficient complementary nucleotides to form base pairs with the antisense region and form a couple. For example, the asymmetric double ANSI molecule of the invention may comprise an antisense region having a length sufficient to mediate the RNAi in a T cell (eg, from about 19 to about 22 (eg, about 19, 20, 21, or 22) nucleotides, and a sense region having from about 3 to about 18 (eg, 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 by "modulated expression of the gene" it is understood that the expression of an objective gene is over-regulated or sub-regulated, which may include the up-regulation or the up-regulation of mRNA levels present in a cell, or the translation of mRNA, or the synthesis of protein or protein subunits, encoded by the target gene The modulation of gene expression can also be determined by the presence, amount or activity of one or more proteins or protein subunits encoded by the protein. target gene that is over-regulated or sub-regulated, of man was that the expression, level or activity of the subject protein or subunit is greater than or less than that observed in the absence of the modulator (e.g., an siRNA). For example, the term "modular" may mean "inhibit", but the use of the word "modular" is not limited to this definition. By "inhibit", "sub-regulate" or "reduce" expression, it is understood that genetic expression or level RNA molecules or equivalent RNA molecules that code for one or more proteins or protein subunits, or the level or activity of one or more proteins or protein subunits encoded by a target gene, are reduces below that observed in the absence of the nucleic acid molecules (eg, ANsi) of the invention. In one embodiment, the inhibition, sub-regulation or reduction with an ANSI molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, the inhibition, sub-regulation or reduction with ANsi molecules is below that level observed in the presence, for example, of an ANSI molecule with mixed sequence or with paired malformations. In another embodiment, the inhibition, sub-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. "Silenced" of the gene refers to the loss of partial or complete function through the directed inhibition of gene expression in a cell and can also be referred to as "deactivation". Depending on the circumstances and the biological problem to be treated, it may be preferable to partially reduce the gene expression. Alternatively, it may be desirable to reduce the genetic 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. O 99/32619. Depending on the analysis, the quantification of the gene expression allows the detection of various degrees of inhibition that may be desirable in certain embodiments of the invention, including prophylactic and therapeutic methods, which will be able to deactivate genetic expression, in terms of the levels of MRNA or levels' or protein activity, for example, equal to or greater than 10%, 30%, 50%, 75%, 90%, 95% or 99% baseline (ie, normal) or other levels of control, including high levels of expression such as can be associated with. particular disease states or other objective conditions for therapy. The phrase "which inhibits the expression of an objective gene" 'refers to the ability of an ANsi of the invention to initiate the silencing of the gene of the target gene. To examine the degree of silencing of the gene, samples or analyzes of the organism of interest, or cells in culture that express a particular construct, are compared to the control samples that lack the expression of the construct. Control samples (which lack construction expression) are assigned a relative value of 100%. Inhibition of the expression of a target gene is achieves when the test value relative to the control is approximately 90%, frequently 50% and in certain modalities 25-0%. Suitable assays include, eg, examination of protein or mRNA levels using techniques known to those of skill in the art such as spot spotting, northern immunochadrosis, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic analyzes. 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 the cells which are themselves subject to the supply of ANsi. "Subject", therefore, can refer to an organism, organ, tissue or cell that includes an organ, tissue or cellular subjects in vivo or ex vivo to which the nucleic acid molecules of the invention can be administered, and be improved by the polynucleotide delivery enhancement polypeptides described herein. Exemplary subjects include individuals or mammalian cells, e.g., patients or human cells. As used herein, "cell" is used in its usual biological sense, and does not refer to a complete multicellular organism, e.g., specifically does not refer to a human. The cell can be present in a organism, e.g., birds, plants, and mammals such as humans, cows, sheep, apes, monkeys, pigs, dogs and cats. The cell can be prokaryotic (e.g., a bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ-line origin, totipotent or pluripotent, dividing or non-dividing.The cell can also be derived from or can comprise a gamete or embryo, a germinal cell or a totally differentiated cell. "vectors" is understood to mean any technique based on 'nucleic acid and / or viral base used to deliver a desired nucleic acid. "By" comprising "is meant to include, but is not limited to, all of the following to the word. "comprising." Therefore, 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. and is limited to, everything following the phrase "consisting of." Therefore, the phrase "consisting of" indicates that the elements listed are required or mandatory and that no other element may be present. essentially "it is understood that it includes any element listed after the phrase, and that it limits other elements that do not interfere with or contribute to the activity or action specified in the description for the items listed. Therefore, the phrase "consisting essentially of" indicates that the items listed are required or 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 at the 2 'position of a residue of beta-D-ribofuranose. 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 of natural origin by addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as to the terminus (s); of ANSI or internally, for example, in one or more RNA nucleotides. The nucleotides in the RNA molecules of the present invention may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleosides or deoxynucleotides. These altered RNAs can be referred to as analogues or RNA analogues of natural origin.

By "highly conserved sequence region" is meant, that a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to another or from one biological system to another. By "sense region" is meant a nucleotide sequence of an ANsi molecule that has complementarity for an antisense region of the ANsi molecule. Additionally, the sense region of an ANsi 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 for a target nucleic acid sequence. Additionally, the antisense region of an ANsi molecule may optionally comprise a nucleic acid sequence having complementarity for a sense region of the ANsi molecule. By "target nucleic acid" is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be a DNA or RNA. By "complementarity" it is meant that a nucleic acid can form hydrogen bond (s) with another nucleic acid sequence by types either Watson- Crick traditional or other non-traditional. With reference to the nucleic acid 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, e.g., RNAi activity, to proceed. The determination of binding-free energies for nucleic acid molecules is well known in the art (see, eg, Turner et al., CSH Symp, Quant.Biol., LII, 1987, pp. 123-133; Frier et al. , Proc. Nati, Acad. Sci., USA 83: 9373-9377, 1986, Turner et al., J. Am. Chem. Soc, 109: 3783-3785, 1987. A percentage complementarity indicates the percentage of waste contiguous in a nucleic acid molecule that can form hydrogen bonds (eg, pairing of Watson-Crick bases) with a second nucleic acid sequence (eg, 5, 6, 7, 8, 9 or 10 nucleotides of a total of 10 nucleotides in the first oligonucleotide that are formed in base pairs to a second nucleic acid sequence, which has 10 nucleotides represents 50%, 60%, 70%, 80%, 90% and 100% complementarity, respectively "Perfectly complementary" means that all contiguous residues of a nucleic acid sequence will be bound by hydrogen with the same of contiguous residues in a second nucleic acid sequence. The term "universal base" as used in the present, refers to nucleotide-based analogues that form base pairs with each of the natural DNA / RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as is known in the art (see, for example, Loakes, Nucleic Acids Research 29: 2437-2447, 2001). The term "acyl nucleotide" as used herein refers to any nucleotide having an acyl ribose sugar, for example, wherein any of the ribose carbons (Cl, C2, C3, C4 or C5) are found independently or in combination, absent from the nucleotide. The term "biodegradable" as used herein, refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation. The term "biologically active molecule" as used herein, refers to compounds or molecules that are capable of emitting or modifying a biological response in a system. Non-limiting examples of biologically active ANSI molecules, either alone or in conjunction with other molecules contemplated by the present invention, include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors,. nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triple-forming oligonucleotides, 2,5-A chimeras, ANsi, dsRNA, allozymes, aptamers, decoys and analogues thereof. the biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and / or the 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 a saturated or unsaturated alkyl group, optionally substituted with OH, COOH,, oxo, amine, or substituted or unsubstituted axyl groups; replaced. By "cap structure" is meant the chemical modifications that have been incorporated into any termination of the oligonucleotide (see, for example, Adamic et al., U.S. Patent No. 5,998,203, incorporated by reference herein). These termination modifications protect the nucleic acid molecule from the Exonuclease degradation, and can help its delivery and / or localization within a cell. The cover may be present at the 5 'end (top 5') or at the 3 'end (top 3') or may be present at both ends. In non-limiting examples, cap 5 'includes, but is not limited to, glyceryl, inverted basic deoxy residue; 4 ', 5'-methylene nucleotide; 1- (beta-D-eritrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1, 5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; nucleotide of modified base; phosphorodithioate linkage; treo-pentofuranosyl nucleotide; acrylic nucleotide 3 ',' -sec; nucleotide of acrylic 3, -dihydroxybutyl; 3.5-dihydroxypentyl acrylic nucleotide; 3 ', 3' -inverted nucleotide residue; abasic residue of 3 ', 3' - ^ inverted; 3 ', 2' -inverted nucleotide residue; basic residue of 2 2 '-inverted; 1,4-butanediol phosphate; .3'-phosphoramidate; hexyl phosphate; aminohexyl phosphate; 3'-phosphate; 3 '-phosphorothioate; phosphorodithioate; or bridge or non-bridge methylphosphonate residue. Non-limiting examples of the 3 'cap include, but are not limited to, glyceryl, inverted deoxy basic residue; 4 ', 5'-methylene nucleotide; nucleotide of 1- (beta-D-eritrofuranosyl), 4'-thio nucleotide; carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6- phosphate aminohexyl; 1, 2-aminododecyl phosphate; hydroxypropyl phosphate; 1, 5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; nucleotide of modified base; phosphorodithioate; treo-pentofuranosyl nucleotide; 3 ', 4' acrylic nucleotide-dry; 3, 4-dihydroxybutyl acrylic nucleotide; 3, 5-dihydroxypentyl nucleotide; 5 ', 5'-inverted nucleotide residue; 5 ', 5'-inverted abasic residue; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5 '-amino; 5'-phosphorusamidate, phosphorothioate and / or bridge and / or non-bridge phosphorodithioate, methylphosphonate and 5'-mercapto bridging or non-bridging (for details see, Beaucage and Lyer, Tetrahedron 49: 1925, 1993 incorporated by 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 substitutions of either sugar and / or phosphate, and allows the bases: The remaining ones exhibit their enzymatic activity. The group or compound is abasic, in that it does not contain a commonly recognized nucleotide base, such as adenosine, cytosine, uracil or thymine and as a consequence lacks a base at position 1 '. By "nucleotide" as used herein, as recognized in the art, includes natural (standard) bases and modified bases well known in the art. such bases are generally located at the 1 'position of a nucleotide sugar residue. The nucleotides generally comprise a base, sugar and a phosphate group. . The nucleotides can be found unmodified or modified in the sugar, phosphate and / or base residue (also referred interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others; see, for example, Usman and McSwigen, supra; Eckstein et al., PCT International Publication No. WO 92/07065; Usman et al., PCT International Publication No. WO 93/15187; Uhlman &Peyman, supra, are all incorporated by reference in the There are several examples of modified nucleic acid bases known in the art as summarized by Limbach, et al., Nucleic Acids Res., 22: 2183, 1994. Some of the non-limiting examples of base modifications that can be introduced in 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, -alkylcytidines (e. g., 5-methylcytidine), 5-alkyluridines (eg, ribotimidine), 5-halouridine (eg, 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (eg, 6-methyluridine), tipping and others (Burgin et al. . , 'Biochemistry 35: 14090, 1996; Uhlman & Peyman, supra). By "modified bases" in this aspect, it is understood nucleotide bases other than adenine, guanine, cytosine and uracil in the 1 'position or their equivalents. By "target site" is meant a sequence within a target RNA that is "targeted" for cleavage mediated by an ANsi construct that contains sequences within the antisense region that are complementary to the target sequence. By "detectable level of cleavage" is meant the unfolding of the target RNA (and the formation of cleaved product RNAs) to a sufficient degree to discern cleavage products above the bottom 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 the material, in a purified or unpurified form, of biological sources, including, but not limited to, human, animal, plant, insect, bacterial, viral or other sources, wherein the system comprises the components required for the 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 in an in vitro configuration.

The term. "Biodegradable linker" as used herein, refers to a nucleic acid or non-nucleic acid binding molecule that is designed as a biodegradable link to connect a molecule to another molecule, for example, a biologically active molecule an ANsi molecule of the invention, or the sense and antisense strands of an ANsi molecule of the invention. The biodegradable bond is designed in such a way 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 binding molecule based on nucleic acid can be modulated using various chemistries, for example, combinations of ribonucleotides, deoxyribonucleotides and chemically modified nucleotides, such as 2'-0-methyl, 2'-fluoro, 2 ' -amino, 2'-0-amino, 2'-C-allyl, 2'-0-allyl, and other 2'-modified or modified base nucleotides. The biodegradable nucleic acid binding molecule can be a dimer, trimer, tetramer or a longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length, or may comprise a single nucleotide with a phosphorus-based bond, for example, a phosphoroamidate or phosphodiester linkage. The biodegradable nucleic acid binding molecule can also understand structural modifications of nucleic acid, nucleic acid sugar or nucleic acid base. By "abasic" is meant sugar residues lacking a base or having other chemical groups in place of a base in the 1 'position, see, for example, Adamic, et al., U.S. Patent. No. 5,998,203. By "unmodified nucleoside" is meant one of the bases of adenine, cytosine, guanine, thymine or uracil bound to the 1 'carbon of beta-D-ribofuranose. By "modified nucleoside" is meant any nucleotide base that contains a modification in the chemical structure of a base, sugar and / or non-modified nucleotide 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 be 2'-NH2 or 2'-0-NH2, which may be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Patent. No. 5,672,695 and Matulic-Adamic et al., U.S. Patent. No. 6,248,878. ANsi molecules can be complexed with cationic lipids, packaged within lipdsomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be administered locally through injection, infusion pump or prosthesis, with or without its incorporation into biopolymers. In another embodiment, polyethylene glycol (PEG) can be covalently bound to the ANsi compounds of the present invention, to the polypeptide that enhances the supply of polynucleotides, or both. The bound PEG can be of any molecular weight, preferably from about 2,000 to about 50,000 Daltons (Da). The sense region can be connected to the antisense region by a linker molecule, such as a polynucleotide linkage or a non-nucleotide linkage. "Inverse repeat" refers to a nucleic acid sequence comprising a sense and an 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 link or a heterologous sequence such as a self-dividing ribozyme between the two elements of the repeat. , The elements of the inverted repeat have a sufficient length to form a double-stranded RNA. Typically, each element of the inverted repeat is from about 15 to about 100 nucleotides in length, preferably from about 20.30 nucleotides in base, preferably from about 20-25 nucleotides in length, eg, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29 or 30 nucleotides in length. "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers in mono or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified structure residues or bonds, which are synthetic, of natural origin and of non-natural origin, which have binding properties similar to those of the nucleic acid of reference, and which are metabolize in a similar way to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphorimidates, methyl phosphonates, chiral methyl phosphonates, 2-0-methyl ribonucleotides, peptide nucleic acids (PNAs). "Large double-stranded RNA" refers to any double-stranded RNA having a size greater than about 40 base pairs (bp) eg, greater than 100 bp or more, particularly greater than 300 bp. The sequence of a large dsRNA can represent a segment of an mRNA or the entire mRNA. The size, maximum of the large dsRNA is not limited in the present. The double-stranded RNA may include modified bases wherein the modification can be made to the phosphate sugar structure or to the nucleoside. Such modifications may include a nitrogen or sulfur heteroatom or any other modification known in the art. The double-stranded structure can be formed by the auto-complementary RNA chain as it occurs for a capillary RNA or a micro RNA or by hybridization of two different complementary RNA strands. "Superimposed" refers to when two RNA fragments have sequences that are superimposed by a plurality of nucleotides on a chain, for example, when the plurality of nucleotides (nt) is as few as 2-5 nucleotides or as 5-10 nucleotides. nucleotides or more. "One or more siRNAs" refers to dsRNAs that differ from each other on the basis of sequence. "Target gene or mRNA" refers to any gene or mRNA of interest. Indeed, any of the genes previously identified by genetics or by sequencing, can represent an objective. The target genes or mRNA may include developmental genes and regulatory genes as well as metabolic or structural genes or genes that encode enzymes. The target gene can be expressed in those cells in which a phenotype or an organism is investigated, 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 vegetable 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 formulas of "a", "an" and "the", "the" include the plural reference unless the context clearly dictates otherwise. EXAMPLES The foregoing description generally describes the present invention, which is further exemplified by the following examples. These examples are described solely for purposes of illustration, and are not intended to limit the scope of the invention. Although specific terms and values have been employed herein, such terms and values will be similarly understood as exemplary and not limiting of the scope of the invention. Example 1 Production and Characterization of Compositions Comprising a siRNA Complexed with a Polypeptide that Enhances the Delivery of Polynucleotides To form complexes between candidate siRNAs and the polynucleotide delivery enhancement polypeptides of the invention, a suitable amount of siRNA is combined with a predetermined amount of the polypeptide that enhances the supply of polynucleotides, for example, in Opti-MEM® cell medium (Invitrogen), in defined ratios and incubated at room temperature for about 10-30 minutes. Subsequently, a selected volume, e.g., approximately 50 μ ?, of this mixture, is contacted with the target cells and the cells are incubated for a predetermined incubation period, which, in the present example was 2 hours. The siRNA / peptide mixture may optionally include a cell culture medium or other additives such as fetal bovine serum. For H3, H4 and H2b, a series of experiments were carried out to complex these polynucleotide supply enhancement polypeptides with siRNA at different ratios. Generally, this started with a ratio of 1: 0.01 to 1:50 of siRNA / histone. To each well in a 96-well microtiter plate, 40 μm of siRNA was added. Each well contained beta-gal cells at a 50% confluence. ' The. Exemplary optimized ratios for transfection efficiency are shown in Table 2 below. The transfections were carried out with either regular siRNA or complexed siRNA. with one of the histone proteins identified above on 9L / beta-gal cells. The siRNA was designed to specifically inactivate mRNA beta-galactosidase, and the activities are expressed as a percentage of beta-gal activities of the control (the control cells were transfected using lipofectamine without the polypeptide that enhances their polynucleotide delivery). . Analyzes to detect and / or to quantify the efficiency of the siRNA supply are carried out using conventional methods, for example, beta-galactosidase analysis or flow cytometry methods. For beta-galactosidase analyzes, 9L / LacZ cells, a cell line constitutively expressing beta-galactosidase, were used. 9L / LacZ cells are rat gliosarcoma fibroblast cells that constitutively express LacZ and were obtained from ATCC C # CRL-2200). 9L / LacZ cells were cultured in Modified Essential Medium of Dulbecco (DMEM) medium with a supplement of 1 mM sodium pyruvate, non-essential amino acids and 20% fetal bovine serum. Cells were cultured at 37 ° C and 5% CO2 supplemented with a mixture of antibiotic containing 100 units / ml penicillin, 100 μ? / P ?? of streptomycin and 0.25 mg / ml of Fungizone (Invitrogen). The siRNA duplex designed against beta-gal mRNA was chemically synthesized and used with delivery reagents to evaluate deactivation efficiency. Peptide Synthesis Peptides were synthesized by solid phase Fmoc chemistry on CLEAR-amide resin using a Rainin Symphony synthesizer. The coupling steps were carried out using 5 equivalents of HCTU and amino acid Fmoc with an excess of N-methylmorpholine for 40 minutes. Removal of Fmoc was achieved by treating the peptide resin with 20% piperidine in DMF for two cycles of 10 minutes Upon completion of the complete peptide, the Fmoc group was removed with piperidine and washed extensively with DMF. Maleimido-modified peptides were prepared by coupling 3.0 equivalents of 3-maleimidopropionic acid and HCTU in the presence of 6 equivalents of N-methylmorpholine to the N-terminus of the peptide resin. The degree of coupling was 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 of triisopropyl silane followed by gentle stirring at room temperature for 2 hours. The resulting crude peptide was collected by trituration with ether followed by filtration. The crude product was dissolved in Millipore water and lyophilized to dryness. The crude peptide was absorbed in 15 ml of water containing 0.05% TFA and 3 ml of acetic acid and loaded onto a reverse phase Zorbax RX-C8 (22 mm ID x 250 mm, particle size 5 μ) to through an injection loop of 5 ml at a flow rate of 5 ml / min. The purification was achieved by passing a linear AB gradient of 0.1% B / min where solvent A is 0.05% TFA in water and solvent B is 0.05% TFA in acetonitrile. The purified peptides were analyzed by HPLC and ESMS. Synthesis and Preparation of siRNA Oligonucleotide synthesis was carried out using the standard method of 2-cyanoethyl phosphoramidite on long chain alkylamine derivatized with pore glass controlled with selected 5-0-dimethyltrityl-2 '-0-t-butyldimethylsilyl-3' -O-succinyl ribonucleoside or support of thymidine 5'-O-dimethyltrityl-2'-deoxy-3'-0-succinol, if applicable. All oligonucleotides were synthesized at either the 0.2 or 1 ymol scale, using an ABI 3400 DNA / RNA synthesized, cleaved from the solid support using concentrated NH4OH, and deprotected using a 3: 1 mixture of NH4OH: EtOH at 55 ° C. Deprotection of the protecting groups of 2'-TBDMS was achieved by incubating the deprotected RNA with base with a solution (600 μ? Per μp) of N-methylpyrrolidone / triethylamine / triethylamine tris (hydrofluoride) (6: 3: 4 by volume) at 65 ° C for 2.5 hours. The corresponding building blocks, 5'-dimethoxytrityl-N- (tac) -2 '-O- (t-butyldimethylsilyl) .- 3' - [(2-cyanoethyl) - (N, N-diisopropyl)] -phosphoramidites of A, U ,. C and G (Proligo, Boulder CO) as well as the modified phosphoramidites, 5 'DMTr-5-methyl-U-TOM-CE-phosphoramidite, 5' -DMTr-2 '-Ome-Ac-C-CE phosphoramidite, 5' -DMTr-2 '-Ome-G-CE phosphoramidite, 5' -DMTr-2 '-Ome-U-CE phosphoramidite, 5' -DMTr-2 '-Ome-A-CE phosphoramidite (Glen Research) were purchased directly from Providers. Triethylamine-trihydrofluoride, N-methylpyrrolidinone and ammonium hydroxide concentrated were purchased from Aldrich. All HPLC analyzes and purifications were carried out in a Waters 2690 with Xterra ™ columns. All other reagents were purchased from Glen Research Inc. The oligonucleotides were purified to more than 97% purity as determined by RP-HPLC. The siRNAs for injection into mice were purchased from Qiagen, which were purified by HPLC after hybridizing to an acceptable endotoxin level for injection in vivo. Primary Human Monocytes: Recent samples of human blood were obtained from healthy donors of Golden West Biologicals. For isolation of monocytes, blood samples were diluted with PBS at a ratio of 1: 1 immediately after receipt. The peripheral blood mononuclear cells (PBMC) were isolated first using the Ficoll gradient (Amersham) of whole blood. Monocytes were further purified from PBMCs using the Miltenyi CD14 positive selection kit and the supplied protocol (MILTENYI BIOTEC). To evaluate the purity of the monocyte preparation, the cells were incubated with an anti-CD14 antibody (BD Biosciences) and then selected by flow cytometry. The purity of the monocyte preparation was greater than 95%. Activation of human monocytes was carried out out adding 0.1-1.0. ng / ml of liposaccharides, LPS (Sigma, St. Louis, MO) to the cell culture to stimulate the production of tumor necrosis factor + (TNF- +). Cells were harvested 3 hours after incubation with LPS and mRNA levels were determined by Quantigene analysis (Genospectra, Fremont, CA) according to the manufacturer's instructions. Mouse Tail Fibroblast Cells Mouse tail fibroblast cells (MTF) were derived from the tails of C57BL / 6J mice. The tails were removed, submerged in 70% ethanol and then cut into small sections with a razor. The sections were washed three times with PBS and then incubated on a shaker at 37 ° C with 0.5 mg / ml collagenase, 100 units / ml penicillin and 100 g / ml streptomycin to rupture the tissue. The tail sections were then cultured in complete medium (Modified Essential Medium from Dulbecco with 20% FBS, 1 mM sodium pyruvate, non-essential amino acids and 100 units / ml penicillin and 100 pg / ml streptomycin) to stabilize the cells Cells were cultured at 37 ° C, 5% CO2 in complete medium as noted above. Transfection Procedure On the first day of the procedure, the saturated 9L / LacZ cultures were taken from the T75 flasks, and the cells were detached and diluted in 10 ml of complete medium (DMEM, 1. x PS, 1 x Na pyruvate, 1 x NEAA). The cells were further diluted, at 1:15, and 100.μ? of this preparation were aliquoted into wells of 96-well plates, which will generally produce approximately 50% cell confluence on the next day for transfection. The edges of the wells are left empty and filled with 250 μ? of water, and the plates are placed non-stacked in the incubator overnight at 37 ° C (incubator with 5% C02). On the second day, the transfection complex is prepared by Opti-MEM, 50 μ? in each well. The medium is removed from the plates, and the wells are washed once with 200 μ? of PBS or Opti-MEM. The plates are stained and completely dried with tissue paper by inversion. The transfection mixture is then added (50 μ? /?) In each well, and 250 μ? of water to the wells on the edge to prevent them from drying out. The cells are then incubated for at least 3 hours at 37 ° C (incubator with 5% CO2). The transfection mixture is removed and replaced with 100 μ? of complete medium (DMEM, 1 x PS, 1 x Na pyruvate, 1 x NEAA). The cells are cultured for a defined length of time and then harvested for enzyme analysis. Cell Viability (MTT Analysis) Cell viability will be evaluated using the MTT analysis (MTT-100, MatTek team). This equipment measures the absorption and transformation of the tetrazolium salt to the formazan dye. A thawed MTT concentrate is prepared and diluted 1 hour before the end of the dosing period with the lipid, mixing 2 ml of the MTT concentrate with 8 ml of MTT diluent. Each cell culture insert is washed twice with PBS containing Ca + 2 and Mg + 2 and then transferred to a new 96-well transport plate containing 100 μ? of the MTT solution mixed per well. This 96-well transport plate is then incubated for 3 hours at 37 ° C and with 5% CO2. After incubation for 3 hours, the MTT solution is removed and the cultures are transferred to a second 96-well feed tray containing 250 μ? of MTT extraction solution by. water well. 150 μ? of an MTT extraction solution to the surface of each culture well and the samples rested at room temperature in the dark for a minimum of '2. hours and maximum of 24 hours. The insert membrane was then punctured with the tip of a pipette and the solutions in the upper and lower wells were allowed to mix. Two hundred microliters of the extracted solution mixed together with the extracted blanks (negative control) were transferred to a 96-well plate for measurement with a microplate reader. The optical density (OD) of the samples was measured at 570 nm with the background subtraction at 650 nm in a plate reader. The Cell viability was expressed as a percentage and calculated by dividing the OD readings for the inserts treated between the OD readings for the inserts treated with PBS and multiplied by 100. For the purposes of this analysis, it was assumed that the PBS had no effect on cell viability and therefore, accounted for 100% of cellular stability. Enzymatic Analysis Reagents for enzyme analysis were purchased from Invitrogen (beta-gal analysis team), and Fisher (Pierce Micro BCA Protein 'Assay Reagent Kit, Catalog). A: Cell lysis Remove the medium, wash once with 200 ul of PBS, stain the dry plate with inversion. Add 30 u'l of lysis buffer of the beta-gal equipment in each well. Freeze-thaw the cells twice to generate the lysate. B: beta-gal analysis Prepare the analysis mixture (50 μ? 1 x buffer, 17 μ? Of ONPG each well) Take a new plate, add 65 μ? of analysis mixture in each well. Add 10 μ? of the cellular lysate in each well.

There must be empty wells. for the subtraction of fund activities. Incubate at 37 ° C for approximately 20 minutes, avoid a long incubation that will use ONPG and tilt the high expression. Add 100 μ? of the detention solution. Measure the OD at 420 nm. C: BCA Analysis Prepare standard BSA (150 μ? Per well), the dots should be doubled on each plate. Place 145 μ? of water inside each well, add 5 μ? of the cellular lysate within each well. Prepare the final analysis reagent according to the manufacturer's instructions. Add 150 μ? of the analysis reagent within each well. Incubate at 37 ° C for approximately 20 minutes. Measure the OD at 562 nm. D: Calculation of the specific activity The specific activity is expressed as nmol of Hydrolysed ONPG / t / mg protein, where t is the incubation time in minutes at 37 ° C; mg protein is the analyzed protein that is determined by the BCA method. Flow Cytometry Measurement of FITC / FAM Conjugate siRNA Fluorescence activated cell (FACS) selection analysis was carried out using a Beckman Coulter FC500 cell analyzer (Fullerton, CA). The instrument was adjusted according to the fluorescence probes used (FAM or Cy5 for siRNA and FITC and PE). for CD14). Propidium iodide (Fluka, St. Louis, O) and Annexin V (R &D Systems, Minneapolis, MN) were used as indicators for cell viability and cytotoxicity. A brief protocol stage by stage is detailed below. a) After exposure to the siRNA / peptide complex, the cells were incubated for at least 3 hours. b) Wash the cells with 200 μ? of PBS. c) Remove the cells with 15 μ? of TE ,, incubate to 37 ° C. d) Resuspend the cells in five wells with 30 μ? of FACS solution (PBS with 0.5% BSA, and 0.1% sodium azide). e) Combine all five wells in a tube. f) Add PI (propidium iodide) 5 μ? inside each tube. g) Analyze the cells with fluorescence activated cell selection (FACS) according to the manufacturer's instructions. The siRNA sequence used to silence the Beta-galactosidase mRNA was as follows: CUACACAAUCAGCGAUÜ.U.DT.DT (SENSE) (SEQ ID NO: 32) AAAUCGCUGAUUUGUGUAGdT.dT (Antisense) (SEQ ID NO: 33) The data for the present example is shown in Table 2. The transfection efficiency is inversely correlated with the amount of beta-galactosidase activity measured from the cell lysate. Upon transfection, a measured decrease in beta-galactosidase activity indicates successful transfection. Therefore, in the absence of transfection, the beta-galactosidase activity measured is 100% and the transfection efficiency is 0%. As beta-galactosidase activity decreases, transfection efficiency increases. For example, in Table 2, the histone H2B plus the siRNA results in a transfection efficiency of 62.03% indicating that the measured beta-galactosidase activity decreased to 37.97%. The same procedure to determine the efficiency of transfection was used for the data presented in Table 3. Table 2: Efficiency of the Delivery of siRNA Mediated by Polypeptides that Improve the Supply of Polynucleotides in 9L / LacZ Cells SiRNA / Peptide / Lipids To evaluate the effects of adding a cationic lipid to a mixture, complex or conjugate of siRNA / polypeptide that improves the polynucleotide delivery, the above procedures were followed except that lipofectamine (Invitrogen) was added to the formulation of siRNA / polynucleotide delivery 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), the siRNA and the peptide were mixed together first in Opti-MEM cell culture medium at room temperature, after which LIPOFECTIN® was added at room temperature to the mixture to form the siRNA / peptide / lipid composition cationic To produce the composition comprised of RVIRVWFQNKRCKDKK (SEQ ID NO: 29), AR si and LIPOFECTIN®, the peptide and LIPOFECTIN® were mixed together first in Opti-MEM cell culture medium, within this mixture was added the SiRNA to form the siRNA / peptide / LI composition POFECTIN®. To produce the composition of SiRNA / peptide / cationic lipid using GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30) or GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31) it does not matter in which order components are added to produce the siRNA / peptide / cationic lipid composition. To produce the siRNA / melittin / LIPOFECTIN®, the siRNA and melittin were first mixed together in Opti-MEM cell culture medium and then LIPOFECTIN® was added to the mixture. To produce the siRNA / histone Hl / LIPOFECTIN® composition, histone Hl and LIPOFECTIN® were first added together in Opti-MEM cell culture medium, mixed thoroughly and then the siRNA was added, mixed completely with the histone LIPOFECTIN® mixture to form the siRNA / histone Hl / LIPOFECTIN® composition. Table 3: Efficiency of the Delivery of siRNA Mediated by Polypeptides for Improvement of Polynucleotide Delivery with and without Cationic Lipid in 9L / LacZ Cells Based on the above results, it is apparent that the polynucleotide supply enhancement polypeptides of the invention can substantially improve the cellular uptake of the siRNAs, while than the addition of an optional cationic lipid to certain, siRNA / polynucleotide supply enhancement polypeptides. of the invention, can substantially improve the efficiency of the siRNA delivery. EXAMPLE 2 Production and Characterization of Compositions Comprising a siRNA Conjugated with a Polypeptide that Enhances the Delivery of TAT-HA Polynucleotides The present example describes the synthesis and absorption activity of specific peptides covalently conjugated to a strand of a siRNA duplex. These conjugates efficiently deliver the siRNA within the cytoplasm. Synthesis of Peptide. The peptides were synthesized by solid phase Fmoc chemistry on CLEAR-amide resin. using a Rainin Symphony synthesizer. The coupling steps were carried out: using 5 equivalents of HCTU and amino acid Fmoc with an excess of N-methylmorpholine for 40 minutes. Removal of Fmoc was achieved by treating the peptide resin with 20% piperidine in DMF for two 10 minute cycles. Upon completion of the complete peptide, the Fmoc group was removed with piperidine and washed extensively with DMF. Maleimido-modified peptides were prepared by coupling 3.0 equivalents of 3- maleimidopropionic and HCTU in the presence of 6 equivalents of N-methylmorpholine at the N-terminus of the peptide resin. The degree of coupling was monitored by the Kaiser test. The peptides were cleaved from the resin by the addition of 10 ml of TFA containing 2.5% water and 2.5 of triisopropyl silane followed by gentle stirring at room temperature for 2 hours. The resulting crude peptide was collected by trituration with ether followed by filtration. The crude product was dissolved in Millipore water and lyophilized until dry. The crude peptide was absorbed in 15 ml of water containing 0.05% TFA and 3 ml of acetic acid and loaded onto a reverse phase Zorbax RX-C8 (22 mm ID x 250 mm, particle size 5 μp?) through a loop, injection of 5 ml at a flow rate of 5 ml / min. The purification was achieved by passing a linear AB gradient of 0.1% B / min where solvent A is 0.05% TFA in water and solvent B is 0.05% TFA in acetonitrile. The purified peptides were analyzed by HPLC and ESMS. Synthesis of Conjugates Both peptides and RNAs are prepared using standard solid phase synthesis methods. The peptide and RNA molecules must be functionalized with specific residues to allow covalent binding to each other. For the peptide, the N-terminal is functionalized, example, with 3-maleimidopropionic acid. However, it is recognized that other functional groups such as bromine or acetyl iodine residues will also function. For the RNA molecule, the 5 'end of the sense chain or the 3' end of the antisense chain is functionalized, for example, with a 1-O-dimethoxytritylyl hexyl disulphide linker according to the following synthetic method . The modified C6SS-5 'oligonucleotide (GCAAGCUGACCCUGAAGUUCAU (SEQ ID NO: - 34); 3, 467 mg; 0.4582 mol) was reduced to the free thiol group with 0.393 mg (3 eq) of tris (2-carboxyethyl) phosphine (TCEP) in 0.3 ml of 0.1 M Triethylamine acetate buffer (TEAA) (pH 7.0) at room temperature for 3 hours. The reduced oligonucleotide was purified by RP HPLC on a Xterra®MS Cie 4.6 × 50 mm column using a linear gradient of 0.30% CH 3 CN in 0.1. of TEAA buffer, pH 7, within 20 minutes (tr = 5.931 min.). The purified reduced oligonucleotide (1361 mg, 0.19085 μp) was dissolved in 0.2 ml of 0.1 M TEAA buffer, pH = 7, and then the peptide with the maleimido residue bound to the N-terminus of the peptide, (0.79 mg, 1.5 eq.) Was added to the oligonucleotide solution. After the addition of the peptide a precipitate immediately formed that disappeared at the addition of 150 μ? of 75% CH3CN / 0.1 from TEAA. After stirring overnight at room temperature environment, the resulting conjugate was purified by RP HPLC on a Xterra® S Cie column of 4.6 x 50 mm using a linear gradient of 0.30% CH3CN in 0.1 M of TEAA buffer, pH 7, within 20 minutes and 100% C within the next 5 minutes (tr = 21,007 min.). the amount of the conjugate was determined by spectrometry based on the molar absorption coefficient calculated at? = 260 nm. The MALDI mass spectrometric analysis showed that the observed peak for the conjugate (10 585.3 amu) | · equals the calculated mass. Yield: 0.509 mg, 0.04815 μp ???, 25.2%. The sense chain of the peptide conjugate and the complementary antisense chain were hybridized in 50 mM potassium acetate, 1 mM magnesium acetate and 15 mM HEPES, pH .7.4 by heating at 90 ° C for 2 minutes, followed. by incubation at 37 ° C for 1 hour The formation of the double-stranded RNA conjugate. it was confirmed by undenatured polyacrylamide gel electrophoresis (15%) followed by staining with ethidium bromide. Structure of the polypeptide-siRNA conjugate (SEQ ID Nos. 34 and 35) Absorption experiments The cells were plated the previous day in 24-well plates. so that they were -50-80% confluent at the time of transference. For the complexes, the siRNA and the peptide were diluted in Opti-MEM® medium (Invitrogen), then mixed and allowed to complex 5-10 minutes before being added to the cells washed with PBS. The final concentration of siRNA was 500 nM at each concentration of peptide (2-50 μ?). The conjugate, also diluted in Opti-MEM medium, was added to the cells at final concentrations ranging from 62.5 nM to 500 nM. At a concentration of 500 nM, it was also combined with 20% FBS just before being added to the washed cells. The cells were transfected for 3 hours at 37 ° C, 5% C02. The cells were washed with PBS, treated with trypsin and then analyzed by "flow cytometry" The uptake of siRNA was measured by the Cy5 fluorescence intensity and the cell viability was evaluated by the addition of propidium iodide. in Figure 1, the peptide / siRNA conjugates achieve greater percent absorption in mouse tail fibroblast cells than the peptide / siRNA complexes In addition, the peptide / siRNA conjugates achieved a higher mean fluorescence intensity (MFI; Figure 2) that the peptide / siRNA complex. data indicate that in certain embodiments, it will be desirable to conjugate the polypeptide that enhances the delivery of polynucleotides to the siRNA molecule. EXAMPLE 3 Display of RNAi Complexes / Delivery Peptide Shows the Efficient Induction of RNAi Absorption in 9L / LacZ Cells by a Different Assembly of Rationally Designed Polynucleotide Delivery Enhancement Polypeptides The present example provides further evidence that a diverse assembly of supply enhancement polypeptides provides the rationally engineered polynucleotide of the invention / improves the absorption of siRNA when complexing with the ARNsis. Approximately 10,000 9L / LacZ cells per well were plated onto 96 well flat bottom plates so that they would be -50% confluent the next day at the time of transfection. The siRNA labeled with FAM and the peptides were diluted in Opti-MEM medium (Invitrogen) at 2 times the final concentration. Equal volumes of siRNA and peptide were mixed and allowed to complex for 5-10 minutes at room temperature and then 50 μ? they were added to the cells, previously washed with PBS. The cells were transfected for 3 hours at 37 ° C, 5% C02. The cells were washed with PBS, treated with trypsin and then analyzed by flow cytometry. . The absorption of siRNA was measured by the FAM fluorescence intensity and the cell viability was evaluated by the addition of propidium iodide. Table 4 below summarizes the cellular uptake percentage data in 9L / LacZ cells for the various rationally engineered polynucleotide delivery enhancement polypeptides. Included in Table 4 is the concentration of the peptide and siRNA used. Table 4: Efficiency of RNAi Absorption Mediated by Rationally Designed Polynucleotide Supply Enhancement Polypeptides in 9L / LacZ Cells EXAMPLE 4 The Provision of siRNA is Enhanced by Polynucleotide Enhancement Polypeptides In Vitro The present example illustrates the enhancement of siRNA uptake by the polynucleotide delivery enhancement polypeptides of the invention in LacZ cells, primary murine fibroblasts and monocytes humans. The materials and methods used for the experiments carried out on 9L / LacZ cells and mouse fibroblast cells are generally the same as described above, except that for murine experiments, 9L / LacZ cells were replaced with mouse tail fibroblasts ( MTF). The materials and. methods used for experiments performed on human monocytes are described below. The results for the transfections carried out with MTF cells are summarized in Table 5. Included in Table 5 is the amino acid sequence of the peptide used and the concentration of the peptide and the Cy5 tag conjugated to the eGFP siRNA. The results for transfections carried out with both MTF and 9L / LacZ cells are summarized in Table 6. The data presented in Table 6 offers a comparison of transfection efficiencies for some Peptide / siRNA complexes in different types of. cells Table 5: Efficiency of RNAi Absorption Mediated by Rationally Designed Polynucleotide Supply Improvement Polypeptides in Murine Tail Fibroblast (MTF) Cells Table 6: Efficiency of RNAi Absorption Mediated by Rationally Designed Polynucleotide Supply Improvement Polypeptides in LacZ Cells and Murine Tail Fibroblast Cells To further characterize the ability of the polynucleotide delivery enhancement polypeptides to transfect cells in culture, human monocytes were incubated with 200 nM of FITC-labeled siRNA complexed with various concentrations of PN73, PN250, PN182, PN58 and PN158. Human monocytes were used in addition to LacZ and mouse fibroblast cells because they are the target cell type in the treatment of rheumatoid arthritis. Fresh human blood samples were acquired from healthy donors of Golden West Biologicals. For the isolation of monocytes, the blood samples were diluted with PBS. at a 1: 1 ratio immediately after receiving them: Peripheral blood mononuclear cells (PBMC) were first isolated by Ficoll gradient (Amersham) from whole blood. The monocytes were further purified from PBMCs using the positive selection Miltenyi CD14 kit and the supplied protocol (MILTENYI BIOTEC). To evaluate the purity of the monocyte preparation, the cells were incubated with an anti-CD14 antibody (BD Biosciences), and then selected by flow cytometry. The purity of the monocyte preparation was greater than 95%. The following description is a brief indication of the transfection protocol used in this example. The cells were plated at a confluence of 70-90% for adhered cell lines and 100,000 cells per well for cells in suspension. For complexes SiRNA / transfection reagent, conjugated siRNA was diluted separately, with FAM 'and peptides in Opti-MEM® medium at 2 times the final concentration. Equal volumes of siRNA and transfection reagent were mixed and allowed to complex 5-10 minutes at room temperature. For siRNA-peptide conjugates, the conjugates were diluted in Opti-MEM® medium directly. The transfection mixtures were added to the cells, previously washed with PBS. The cells were transfected for 3 hours at 37 ° C, 5% C02. For the analysis of siRNA uptake, the cells were washed with PBS, treated with trypsin (adherent cells only) and then analyzed by flow cytometry. The uptake of siRNA was measured by the intensity of intracellular Cy5 or FAM fluorescence. Cell viability was determined using propidium iodide (absorption) or Annexin V-PE (staining). The above experiment shows that the polypeptide that enhances the exemplary polynucleotide delivery, PN73 is a . ideal candidate for the treatment of rheumatoid arthritis. Figure 3 illustrates the ability of several different polynucleotide delivery enhancement polypeptides to improve the absorption of siRNA in human monocytes in culture. Transfection with lipofectamine was used as a comparison. Cell viability was also established for each peptide (Figure 4). The data show the surprising and unexpected discovery that peptide PN73 transfects human monocytes with high efficiency and low toxicity indicating that it is an ideal candidate for the treatment of rheumatoid arthritis in vivo. EXAMPLE -5 The Delivery of siRNA is Improved by the Conjugation of siRNA to Polynucleotide Supply Improvement Polypeptides The present example provides results from. The visualizations to evaluate the activity of siRNA / polypeptide conjugates that improves the supply of polynucleotides to induce or enhance the uptake of siRNA in 9L / LacZ cell lines and primary mouse stem fibroblast. The materials and methods employed for these studies are generally the same as described above, except that siRNA / peptide mixing is not required as is necessary to produce siRNA / peptide complexes. The Percent absorption for the transfers made with 9L / LacZ cells are summarized in Table 7. Included in Table 7 is the peptide used and the concentration of the peptide / siRNA conjugate. A FAB-b-gal label conjugated to the siRNA molecule was used. The results for the transferences made with MTF are summarized in Table 8. Included in Table 8 is the concentration of the peptide / siRNA conjugate used and the Cy5 label conjugated to the siRNA molecule eGFP. Table 7: Efficiency of RNAi Absorption Mediated by Rationally Designed Polynucleotide Supply Improvement Polypeptides Conjugated to Laczy Cells in LacZ Table 8: Efficiency of RNAi Absorption Mediated by Rationally Designed Polynucleotide Supply Improvement Polypeptides Conjugated to RNAse in Murine Tail Fibroblast Cells The above data show that a diverse assembly of siRNA / peptide conjugates of the invention mediates the delivery of the siRNAs within different cell types at high efficiency. EXAMPLE 6 Deactivation of siRNA Gene Expression Enhanced by Enhanced Polynucleotide Delivery Enhancing Polypeptides to siRNA The present example demonstrates the effective deactivation of target gene expression by siRNA / polypeptide complexes that enhances the polynucleotide supply of the invention. In current studies, the ability of peptide / siRNA complexes to modulate the expression of a human tumor necrosis factor-a gene (hTNF-a), mediated the occurrence or progress of RA upon overexpression in human subjects, was tested and other mammals. Healthy human blood was purchased from Golden West Biologicals (CA), peripheral blood mononuclear cells (PBMC) were purified from the blood using a Ficoll-Pague plus gradient (Amersham). Human monocytes were then purified from the fraction of PBMCs using magnetic microbeads from Miltenyi Biotech. The isolated human monocytes were resuspended in IMDM supplemented with 4 mM glutamine, 10% FBS, 1 x non-essential amino acid and 1 x pen-strep, and stored at 4 ° C until use. In a 96-well round-bottom plate, human monocytes were cultured at 10,000 per well, per 100 μ? in Opti-MEM medium (Invitrogen). The transfection reagent was mixed with the siRNA at the desired concentration in Opti-MEM medium at room temperature for 20 minutes (for Lipofectamine 2000; Invitrogen), or 5 minutes (for peptide). At the end of the incubation, FBS was added to the mixture (final 3%), and 50 μ? of the mixture to the cells. The cells were incubated at 37 ° C for 3 hours. After transfection, the cells were transferred to a V-bottom plate and the cells were pelleted at 1500 rpm for 5 minutes. The cells were resuspended in growth medium (IMDM with glutamine, non-essential amino acid and pen-strep). After overnight incubation, the cells were stimulated with LPS (sigma) at 1 ng / ml for 3 hours. After induction, the cells are collected as above for the quantification of mRNA, and the supernatant was saved for protein quantification. For the mRNA measurement, branched DNA technology from Genospectra (CA) was used according to the manufacturer's specification. To quantify the level of mRNA in the cells, both the guardian gene (cypB) and the mRNA of the target gene (TNF-) were measured, and the reading for TNF-oc was normalized with cypB to obtain a unit of relative luminescence . To quantify the level of proteins, TNF-a ELISA from BD Bioscience was used according to the manufacturer's specification. The siRNAs were targeted to different target regions of the TNF-α mRNA as illustrated in Table 9 below. Not shown for each oligo listed in Table 9 are the 3 'overhangs (e.g., dNdN where N represents any nucleotide). Table 9: Nomenclature, Location of the Target Sequence in the TNF-OI Gene and Oligo Sequence for the siRNA Direction of TNF-a Tables 10, 11 and 12 illustrate the effectiveness of specific oligos complexed to a polypeptide that enhances the supply of polynucleotides of the invention to direct and inactivate the levels of genetic expression of TNF- in human monocytes.

Table 10: Percentage of TNF Deactivation - Mediated by a PN73 / ARNSI Complex Table 11: Percentage of TNF-a Deactivation Mediated by a Complex of PN509 / siRNA Table 12: Percentage of TNF-a Deactivation Mediated by a PN250 / siRNA Complex The above results (Tables 10, 11 and 12) show that effective levels of the inactivation of TNF- gene expression can be achieved in mammalian cells using the new siRNA / polypeptide compositions - which improves the supply of polynucleotides of the invention . Visualization and Characterization Human monocytes (CD14 +) treated with LPS induce the TNF-OI specific mRNA within about 2 hours, followed by peak levels of TNF-α protein 2 hours later. The siRNAs were visualized by deactivation activity by transferring the monocytes with siRNA candidate sequences using Lipofectamine 2000, treating the LPS-infected cells, and measuring TNF-α mRNA levels approximately 16 hours later. Fifty-six siRNA sequences were designed and visualized for their ability to inactivate TNF-a mRNA and protein levels in activated primary human monocytes. The activities for a representative set of 27 siRNA sequences varied from 80% ARMm deactivation activity to no detectable activity. in general, TNF-α protein levels were reduced more than mRNA levels, eg, a 50% deactivation in TNF-α (TNF-a 1) mRNA resulted in a 75% reduction in the level of TNF-a protein. The dose response curves for selected siRNAs that exhibited deactivation levels of 30% to 60%. The calculated IC50 values were found in the range of 10 pMolar to 200 pMolar. Although the siRNA sequences evaluated were distributed throughout the transcription of TNF-OI, the most potent siRNAs identified were located in two areas: half of the coding region and in the 3'-UTR. EXAMPLE 7 Deactivation of siRNA Gene Expression Improves by Delivery Enhancement Polypeptides of Polynucleotide Conjugated with siRNA The present example demonstrates the deactivation of the target gene expression by the peptide-siRNA conjugates of the invention. The materials and methods for these studies are the same as those described above, with the exception that no mixing of the siRNA and the peptide is required. In the present series of studies, deactivation experiments included the comparison of peptide / siRNA-mediated deactivation with and without lipofectamine. The results of this example are illustrated in Table 13 below. Table 13: Peptide / siRNA-Mediated Deactivation of the Gene Expression of TNF- with and without Lipofectamine- The above data (Table 13) evidence that a diverse assembly of the polynucleotide delivery enhancement polypeptides of the invention conjugated to siRNAs functions to enhance siRNA-mediated deactivation of TNF-α gene expression in mammalian subjects. EXAMPLE 8 Time Course of Deactivation of siRNA Gene Expression The present example presents studies concerning the time course of deactivation of siRNA-mediated gene expression. To test the duration of the siRNA effect, the siRNA transfection procedures were employed as noted above, except that the fibroblasts derived from mice expressing eGFP were used. The transfection reagent used here was Lipofectamine. The cells were re-plated on day 18 due to their overgrowth. The second transfection was carried out on day 19, after the first transfection. On day 19, eGFP levels were measured after transfection. Mixed or non-sense siRNA (Qiagen) was used as control, together with GFPI siRNA (GFPI) and a capillary siRNA (D # 21). The deactivation activities were calibrated with mixed siRNA (Qiagen control). A higher value indicates greater deactivation activity.

Table 14: Time Course of Deactivation of TNF- Gene Expression by Transfection. Mediated by Lipofectamine of siRNA Previous studies (Table 14) show that the deactivation activity of the siRNA became apparent around day 3, and was sustained until day 9, after which the target gene expression returned to baseline levels around day 17. After the second transfection on day 18, there was another -reduction of eGFP expression indicating that the reagent can be repeatedly administered to the cells to produce a repeated or lasting deactivation of the gene expression. EXAMPLE 9 Dose Dependence Dependency of TNF-g Gene Expression Mediated by siRNA Complexed with Polypeptide Enhancing Polynucleotide Delivery The present example demonstrates that siRNA-mediated deactivation activity complexed with a polypeptide that improves The provision of polynucleotides, PN73, in activated human monocytes, depends on the dose concentration of the peptide-siRNA complex. The PN73 / siRNA complex was provided at a constant ratio between PN73 and siRNA of approximately PN73: siRNA = 82: 1 (Table 15). Nanomolar siRNA of four hundred complexed with 33 μ? of PN73 for 5 minutes in OptiMEM medium. After complexation, the complex was serially diluted (1: 2 ratio) with OptiMEM. The complex was added to human monocytes for transfection. The following induction and quantification of mRNA was carried out according to the above description.

Table 15: Dependence on Peptide Dosage of Deactivation of TNF-a Gene Expression In a related series of experiments, the RNAs were serially diluted and combined with a fixed amount of PN73 (1.67 μ?). Alternatively described, the polypeptide enhancing the PN73 polynucleotide delivery was complexed with titre amounts of siRNA. The PN73 (1.67 μ?) Was complexed with each LC20 siRNA titration amount for 5 minutes at room temperature in OptiME medium. After complexation, the complex was added to human monocytes for transfection. The induction and quantification data of mRNA provided in Table 16, below, were obtained by the methods described above. Table 16: Dependence on the siRNA dose of the Deactivation of TNF-a Gene Expression EXAMPLE 10 Multiple Dosage Protocol for Extending the Effect of Deactivation of siRNA in Mammalian Cells The present example demonstrates that multiple dosage programs will effectively extend the effects of deactivation of gene expression in mammalian cells, mediated by siRNA / compositions. polypeptide 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 transitions were conducted at the indicated times. The mixed siRNA (Qiagen) was used for side-by-side controls. Table 17 summarizes the data for multiple transitions with a peptide / siRNA complex. The percent deactivation activity of the TNF-a gene represents the percentage of the total gene expression.

Table 17: Deactivation Activity of TNF-a Gene Expression After Multiple Transitions with a Peptide / siRNA Complex The previous results (Table 17) show that when multiple transitions are made in time (in this case between approximately 5 ° -7 ° day after the first transfection), the effects of the deactivation of TNF-a gene expression in mammalian cells can be maintained or re-induced. EXAMPLE 11 Activity of Deactivation of TNF-α Gene Expression Mediated by In Vivo siRNA / Peptide The present example provides in vivo data demonstrating the efficacy of siRNA / polypeptide compositions that enhances the polynucleotide delivery of the invention, to mediate the systemic supply and deactivation of the therapeutic gene through siRNA, effective to modulate the objective gene expression and to modify the phenotype of the cells in a therapeutic manner. Mice expressing human TNF-α were purchased from Hellenic Pasture Institute, Greece, at 5 weeks of age. The mice were administered by intravenous (IV) 300 μ? of saline twice a week (4 mice), the drug for RA Ramicade (5 mg / kg) once a week (2 mice), or siRNA LC20 (2 mg / kg) mixed with PN73 at a molar ratio of 1: 5 twice a week (2 mice). During the injection periods, plasma samples were collected for ELISA test (R & D Systems), and leg tags were taken twice a week as an accepted index of RA disease progression and therapeutic efficacy. The blood plasma levels of the TNF-α protein of the treated mice are shown below in Table 18.

. | Table 18: Amount of TNF-a Protein in Blood Plasma Analyzed by ELISA * These data represent the average of the mice in the experiment in pg / ml. The above results demonstrate the effective reduction of TNF-protein levels in mice treated with peptide / siRNA in circulating blood compared to levels in mice treated with Ramicade or saline (control). Further evidence was obtained of the in vivo efficacy of the siRNA / polypeptide compositions which improves the delivery of polynucleotides and methods of the invention, from the previous murine subjects using foot tags, an accepted phenotypic index for the RA disease state and the efficacy of the treatment. Due to the difference in the starting point (some animals present with marks at earlier points), the marks were adjusted to 0 for all animals in the experiments. Each leg is given a mark between 0 and 3, with the highest mark of 12, according to the following marking index. 0: normal 1: edema or distortion of the leg or ankle joints. 2: distortion of the leg or ankle joints. 3: ankylosis of the wrist or ankle joints.

The results of these footmark evaluations are presented graphically in Figure 5. The data demonstrates that the polypeptide. which improves the PN73 polynucleotide delivery, can deliver therapeutic amounts of siRNA (eg, LC20, TNF-a2 and TNF-OI9 <(UAGCCCAÜGUUGUAGCAAA (SEQ ID NO: 187))) when injected into animals as shown by a delayed progression of RA at week 8. Mice treated with PN73 / siRNA scored better on the paw-marking index at week 8 compared to mice treated with Ramicade. When the paw mark evaluations were carried out at 11 weeks post-treatment, the PN73 / LC20 complex achieved paw tag evaluations comparable to those of mice treated with Ramicade. At a ratio of 1: 5 for peptide PN73 / siRNA LC20, 2 mg / kg LC20 achieved the greatest relative delay observed in the progression of RA compared to the lower doses of LC20 tested. Table 19 below summarizes the relative effectiveness of various siRNAs for 5 different groups evaluated after treatment with PN73 and siRNA.

Table 19: Summaries by Group * The ARNsis were tested in the presence or absence of PN73; Ramicade is a positive treatment control; PBS is a negative treatment control. The above results demonstrate that the siRNA and polypeptide compositions that enhance the polynucleotide delivery of the invention provide promising new therapeutic tools for regulating gene expression and for treating and managing diseases. The siRNAs of the invention, for example the siRNAs that direct TNF-specific mRNAs for degradation, offer greater specificity, lower immunogenicity and greater modification of the disease than current small molecule therapies, soluble receptor or antibody for RA. HE. visualized more than 50 siRNA candidate sequences that target hTNF-oi and produced single administration inactivations of 30 to 85%. More than 20 peptide complexes designed in silico and / or covalent molecules were compared by absorption of fluorescent RNA by monocytes, and a number was found to have a significantly better absorption than the siRNA conjugated to lipofectamine or cholesterol and with values of < 10 pM ICso- The peptide-siRNA formulations efficiently deactivated the levels of TNF-α and protein mRNA in human monocytes activated in vitro. An exemplary candidate peptide / siRNA delivery formulation was evaluated in two transgenic mouse models of rheumatoid arthritis (RA) that constitutively express human TNF-OI. Animals treated with 2 mg / kg of siRNA by IV injection or infliximab twice a week starting at the age of 6 weeks, showed a stabilization of the RA score (leg and joint inflammation) starting at the age of 7 weeks , compared to controls where these disease conditions persisted until week 10. At the age of 9 weeks, animals treated with siRNA showed comparable reductions in RA markings, but plasma TNF-a protein levels significantly less than animals treated with infliximab.

Based on the description herein, the use of siRNA to inhibit the expression of target genes, for example cytosines such as TNF-α, which play important roles in disease states, such as inflammation, provides effective treatments for alleviating or preventing disease symptoms, exemplified by RA, in mammalian subjects. Exemplary peptide / siRNA compositions employed within the methods and compositions of the invention provide advantages related to their ability to reduce or eliminate the target gene expression, eg, expression of TNF-, rather than complexing with the gene product. target, eg, TNF-, 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 siRNAs as drugs. The specific challenges in this context include the delivery through tissue barriers to a target cell or tissue, maintaining the stability of the siRNA, and the intracellular supply of siRNA through cell membranes 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 direct specific gene expression, such as TNF- expression.

I heard that it attenuates disease activity in transgenic animal models predictive of target diseases, as exemplified by studies using murine RA models. In related studies, the compositions and methods of the invention effectively inhibit the expression of TNF-OI in activated monocytes derived from patients with RA. These results indicate that the RNAi pathway effectively mediates the alteration of the cellular phenotype and the progression of the disease through. intracellular effects on TNF trajectories, and avoids the effects of toxicity due to circulating complexes of antibody / TNF-a with the residual immunoreactivity that characterizes current antibody therapies for RA. Notoriously, all tests herein were implemented with minimized associated toxicity effects, such that the doses of ANsis and polynucleotide supply enhancement polypeptides shown in these examples always correlated with cell viability levels of at least 80 -90% or greater. EXAMPLE 12 Optimization of the Rational Design of Polynucleotide Supply Improvement Polypeptides The present example provides an exemplary design and data for optimizing the polynucleotide delivery enhancement polypeptides of the invention. The manipulations of the subject rational design were conducted for a polypeptide that improves the delivery of polynucleotides derived from histone H2B. Table 20: Deletion and Modification of PN73 Table 20 provides a diagram of the primary structure of PN73 and its derivatives generated to optimize the rational design of polynucleotide delivery enhancement polypeptides based on PN73. The gray terminal C of each peptide represents the hydrophobic domain of the peptide and the Black N-terminus of each peptide represents the hydrophilic domain. It was previously shown that the present PN73 peptide is an example of a polypeptide that enhances the delivery of polynucleotides to induce or enhance the delivery of siRNA to cells.

In order to better understand the structural function-activity relationships of these and other polynucleotide supply enhancement polypeptides, primary structural studies were carried out characterizing the C and N terminal function and the activity of conjugates between PN73 and other chemical residues. . The amino acid sequence for the human protein histone 2B (H2B) is shown below. PN73, PN360 and PN361 are fragments of H2B peptide and the portion of the H2B protein they represent is identified below in parentheses after the name of the peptide. The amino acid sequence for PN360 and PN361 listed below is aligned with the corresponding amino acid sequence found in PN73. The PN73 peptide fragment is underlined in the amino acid sequence of H2B and represents the amino acids of H2B 13 to 48. It can also be represented by the amino acids of H2B 12 to 48. The PN360 shares the N terminal with PN73 but lacks the terminal C of PN73 while PN361 shares terminal C with PN73 but lacks terminal N of PN73. The PN73 conjugate is PN73 covalently linked to a single strand of siRNA (e.g., the sense strand). PN404 is a version of PN73 in which all lysines are replaced with arginines and PN509 is a pegylated PN73 (molecular weight of PEG 1K Dalton) derivative found pegylated at the N terminal amino acid sequence H2B (history 2B) PEPAKSAPAPKKGSKKAVTKAQKKDSKKRKRSRKESYSVYVYKVLKVHPD TGI SSKAMGIMNSFVNDI FERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVS EGTKAVTKYTSSK (SEQ ID NO: 164) PN73 (13-48) NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 59) P 360 (13-35; N-terminus of PN73) NH2-KGSKKAVTKAQKKDGKKRKRSRK-amide (SEQ ID NO: 57) PN361 (24-48; C-terminus of PN73) • NH2-KKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO : 58) Conjugates of FN73 (13-48) -ARNsi (sense chain) siRNA-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 59) PN404 (PN73 where all lysines are replaced with arginines) NH2-RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ-amide (SEQ ID NO: 91) PN509 (PN73 pegylated) PEG-RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ-amide "(SEQ ID NO: 90). Figure 6 provides the results of absorption efficiency and cell viability studies of mouse tail fibroblast for rationally engineered polynucleotide enhancement polypeptides PN73 above. The changes in the activity of modified PN73 in mouse tail fibroblast cells are illustrated. Unlike PN404, PN509 increases absorption without increasing toxicity. Although the deletion of part of the terminal of PN73 reduces the activity, the removal of the residues of terminal C eliminates the activity. Both PN73 and PN509 show higher activity in primary cells than Lipofectamine (Invitrogen, CA). EXAMPLE 13 The Polypeptide that Enhances the Delivery of Acetylated Polynucleotides Has Increased Plasma Stability The purpose of the present example was to determine whether modification of the polypeptide that improves the PN73 polynucleotide delivery would provide increased stability to the peptide and consequently improve its transferase activity. The stability of the pegylated N-terminal and acetylated N-terminal PN73 forms in plasma was compared. Terminal C of PN73 is amidated. Size exclusion chromatography with an ultraviolet detector was used to characterize the stability of the unmodified and modified forms of the PN73 before and after its incubation in plasma.

In the absence of plasma, the pegylated and acetylated non-modified forms of PN73 showed distinct UV traces but nevertheless superimposed at approximately 9 minutes. However, after 4 hours of plasma exposure, the UV traces specific for PN73 unmodified and PN73 pegylated were not already present indicating significant degradation. In contrast, the distinct UV trace for the acetylated PN73 remained, indicating that this modification. significantly increased the stability of the PN73 in plasmid compared to the non-modified and pegylated forms of PN73. These data show the surprising and unexpected discovery that the stability of PN73 in plasma can be improved by acetylation of the N-terminus of PN7.3 peptide. The primary structure of the acetylated PN73 peptide is as follows: Ac-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 59) EXAMPLE 14 Polypeptide that improves the supply of polynucleotides does not emit an Interferon Response The purpose of the present example- was to compare the interferon response of transfected cells with either liposomal reagent plus siRNA or with the polypeptide improving the supply of polynucleotides, PN73 peptide plus siRNA. The Responsiveness to interferon was analyzed by ELISA (protein) and ADNb (mRNA levels). Traditionally, siRNA molecules are delivered into cells by liposomal mediated transfection. However, this typically results in low delivery efficiency, an inflammatory response in vivo and an over-regulation of interferon gene expression that results in an inhibition of cell growth. Consequently, there is a limited reduction in the levels of target gene expression, making the siRNA an ineffective method and treatment tool for the study of gene expression. The supply of the siRNA by PN73 solves this problem. Figure 7 provides the results of the bDNA analysis of. lipofectamine aga transfection peptide PN73 of several different siRNAs. The siRNA forms complejoron either with lipofectamine or with PN73 at concentrations of 1 nM, 10 nM, 100 nM or 200 nM. Interleukin 1 beta (IL-? ß) served as a molecular marker to determine the responsiveness of interferon and Qneg was used as a negative control. As shown in Figure 7, lipofectamine complexed 100 nM or 200 nM of TNF-OI siRNA caused a significant increase in mRNA levels IL-? ß. In addition, all of the other tested siRNAs caused a slight increase in IL-β-mRNA levels. In contrast, the same siRNAs complexed with the PN73 peptide did not cause an increase in IL-β-mRNA levels. To further characterize the difference in the response capacity of the interferon observed between cells transfected with either lipofectamine and transfection of PN73, an ELISA was carried out to determine the protein expression levels of the following molecular markers: interleukin 1β (IL -? ß), interferon-a (TNF-a), interferon- (INF-), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-12 (IL-12), MIP- l, Interferon-y (IFN-y), and Tumor Necrosis Factor-a (TNF-a). Table 21 summarizes the relative levels of protein expression of cells transfected with lipofectamine complexed with siRNA or PN73 complexed with siRNA.

Table 21: Relative Levels of Protein Expression of Molecular Markers of the Response Capacity of. Interferon As shown in Table 21, both LC20 and LC17 siRNA had no interferon response regardless of which transfection reagent was used. However, the transfection of IFN-1 or TNF-a9 with lipofectamine caused an increase in the expression levels of IL-ββ, IL-6 and MIP-la proteins. In contrast, the transfection of all the siRNAs tested with PN73 did not cause an observable induction of protein expression in any of the interferon response markers tested.

These data from the ELISA analysis show the surprising and unexpected discovery that PN73-mediated transfection of ARNsis does not emit an interferon response. EXAMPLE 15 The siRNA conjugated with a polypeptide that improves the supply of polynucleotides Provides greater deactivation activity than the siRNA complexed with polypeptide that improves the supply of polynucleotides The purpose of the present example was to compare the deactivation activities in human nymphs of the LC13 siRNAs and LC20 either conjugated or complexed with the polypeptide that enhances the exemplary PN73 polynucleotide delivery. The isolation and transfection of human chiggers as well as the methods used to measure deactivation activity were discussed above. Qneg represents a random siRNA sequence and functions as the negative control in these experiments. The Qneg deactivation activity observed is normalized to 100% (levels of gene expression at 100%) and the activity of LC20 and LC13 is presented as a relative percentage of the negative control. LC20 and LC13 are siRNAs directed against the human TNF-gene. Figure 8 shows the deactivation activity for the LC20 and LC13 siRNAs without PN73 (Figure 8-C), complexed with PN73 (Figure 8-B) or conjugates with PN73 (Figure 8-A) .. LC20 and LC13 were tested over a concentration range of 0 nM to 2.5 nM. PN73 was maintained at a 1: 1 ratio in both the complex and conjugate experiments. As expected, in the absence of PN73, the LC13 and the LC20 showed little deactivation activity (Figure 8-C). Both LC13 and LC20 caused a decrease of approximately 15% and 30% in the genetic expression of TNF-a in relation to the control of Qrieg when complexed with PN73 (Figure 8-B). However, the deactivation activity for TNF-α was reduced to below 60% when the siRNA was conjugated to PN73 (Figure 8-A). This is a significant increase in siRNA deactivation activity compared to the PN73 / siRNA complex. Therefore, these data show the surprising and unexpected discovery that the siRNA deactivation activity is significantly improved when the siRNA is conjugated with the polypeptide that enhances the delivery of exemplary PN73 polynucleotides. EXAMPLE 16 Serum Inhibition of Absorption of Improved siRNA with cholesterol is rescued by a polypeptide that improves the supply of polynucleotides The present example demonstrates that the addition of a permeabilization peptide to the delivery formulation comprising an siRNA conjugated to a cholesterol residue, reduces the inhibitory effects of serum on the absorption of cholesterol-siRNA in a dose-dependent manner. For the analysis of siRNA absorption, the. cells were washed with PBS, treated with trypsin (only bound cells), and then analyzed by flow cytometry. The uptake of the designated RNA, BA, described above, was also measured by the intensity. of Cy5 or FAM fluorescence in the cells, and cell viability was assessed by the addition of 'propidium iodide or AnnexinV-PE. In order to differentiate the cellular uptake of the membrane insert from the fluorescently labeled siRNA, trypan blue was used to saturate the fluorescence on the surface of the cell membrane. Table 22: PN73 Mediated Transfection Rescues the Serum Induced Inhibition of Cellular Absorption of the Cholesterol-conjugated siRNA as Analyzed by Medium Fluorescent Intensity (MFI) The data in Table 22 show that the presence of serum significantly reduces the cellular uptake of the conjugated siRNA to a cholesterol residue according to the invention. However, the cellular uptake of the unconjugated siRNA is rescued in the presence of an exemplary PN73 delivery enhancement peptide. A comparison of the cellular uptake of the conjugated Arnsi with cholesterol according to the invention was carried out, in complex with a permeabilization peptide supply enhancing agent, PN73 (cholesterol siRNA + PN73), and in a non-conjugated siRNA in complex with PN73 (siRNA + PN.73). As shown in Figure 9, the equivalent cellular absorption activity can be achieved at high concentrations of PN73 either with unconjugated siRNA or with siRNA conjugated with cholesterol. For these and related absorption analyzes, cholesterol conjugated siRNA and siRNA / PN73 complex were transfected into mouse tail fibroblast (MTF) cells in Opti-MEM® medium (Invitrogen) as described above, with added serum and concentrations fixed or varied). The final concentration of the siRNA for both cholesterol and complex was 0.2 μ ?. The absorption efficiency and the mean intensity of fluorescence were evaluated by flow cytometry. The ability of PN73 and additionally of PN250 to rescue the inhibition in serum of cell absorption of the. Cholesterol-conjugated siRNA was further characterized in mouse tail fibroblast (MTF) cells. Figure 10 illustrates the effect of the increase in the concentration of fetal bovine serum (PBS) on the cellular uptake of siRNA. In the absence of either PN73 or PN250, the cellular uptake of the cholesterol-conjugated siRNA drastically compromises only 5% of FES. "However, in the presence of either 40 μ? Of PN73 or 80 μ? Of PN250, The absorption of the siRNA is rescued The previous data (Table 22 and Figure 10) show that the conjugation with cholesterol of the Arnsis can significantly improve its cellular absorption, however, the absorption of cholesterol-conjugated siRNAs can be substantially reduced or even eliminated. Because of the presence of serum, this is likely due to the binding of the cholesterol residue with serum proteins that inhibit the ability of the siRNAs attached to cholesterol to enter the target cells, however, in the presence of a supply enhancement agent. , exemplified by the permeabilization peptides PN73 and PN250, the inhibition in serum of cell absorption can be rescued, more specifically, the addition of a peptide. of permeabilization to a delivery formulation comprising an siRNA conjugated to a cholesterol residue, reduces the inhibitory effects of serum on the absorption of cholesterol-siRNA from way dependent on the dose. EXAMPLE 17 Suppression Analysis of the Polypeptide that Enhances the Delivery of Polynucleotides Exemplary The present example illustrates the experimental design employed to optimize the cellular uptake of the siRNA and the deactivation activities of the target gene mediated by siRNA of the polypeptide enhancing the PN73 polynucleotide delivery exemplary . Table 23, below, shows the polypeptide that enhances the delivery of exemplary PN73 polynucleotides and their truncated derivatives. The amino acid sequence for PN360 and PN361 listed below is aligned with the corresponding amino acid sequence of PN73. The PN360 shares its N terminal with PN73 but lacks the C terminal of PN73 while the PN361 shares its terminal C with PN73 but lacks the N terminal of PN73. PN766 represents the 15 amino acids of terminal C of PN73. PN73, PN360, PN361 and PN766 are not labeled with a FITC at terminal C (fluorescence-5-isothiocyanate) (i.e., -GK [EPSILON] G-amide). Table 23 further shows the 11 truncated forms of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides that were created by sequentially deleting 3 residues at a time, except PN768, from the N-terminus of the peptide. All these peptides were labeled with a FITC mark at terminal C (fluorescence-5-isothiocyanate) (i.e., GK [EPSILON] G-amide) so that the cells containing the peptide could be detected by fluorescent microscopy and / or stored by flow cytometry. As a note, PN766 and PN708 have the same amino acid sequence but differ in that PN708 has the FITC label in terminal C. below; is an explanation of the primary structure of PN73 and the truncated forms that will be examined for their transfection activity.

Table 23: PN73 Suppression Series EXAMPLE 18 Suppression Analysis of the Polypeptide that Enhances the Delivery of Polynucleotides Exemplary The functional domains of the polypeptide that the delivery of exemplary PN73 polynucleotides are critical for the ability of the polypeptide to enhance the delivery of polynucleotides to efficiently deliver the ANsis within the cells. These functional domains include membrane binding to fusogenic and nucleotide binding regions. Briefly, membrane binding describes the ability of the polypeptide to improve the delivery of exemplary polynucleotides to bind the cell membrane. The fusogenic character reflects a capacity to detach from the cell membrane and enter the cytoplasm. The membrane binding and the fusogenic domains of the peptide are closely linked mechanically (i.e., the ability of the peptides to enter the cell) and therefore can be difficult to differentiate experimentally. Consequently, these two properties (peptide domains) will be combined in a single analysis. Finally, the nucleotide binding describes the ability of the peptide to bind to nucleotides. The procedures taken to define each of the above-described domains of the polypeptide that improves the polynucleotide delivery are discussed in more detail below. Table 24 summarizes the data for defining the membrane / fusogenic and nucleotide binding domains of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides (data for all concentrations tested are not shown).

Table 24: Summary of the Functional Domain Analysis of the Peptide Suppression Series. PN73 NT = not tested; the peptide concentrations (in parentheses) given are those that achieved absorption given, in percentages, or MFI in relative values. Membrane Binding and Fusogenic Domain of the Exemplary Polynucleotide Enhancement Polypeptide: The efficacy of the full length and truncated forms of the polypeptide that enhances the delivery of exemplary PN73 polynucleotide to be introduced into the cells was tested in vitro by a cellular absorption analysis with primary mouse tail fibroblast cells (MTF). The number of cells in culture that receive the FITC-labeled peptide was measured by flow cytometry. The percentage of cellular absorption of the peptide was expressed in relation to the total number. of cells present in the culture. Additionally, fluorescent mean intensity (MFI) was used to evaluate the amount of FITC-labeled peptide found within the cells. The MFI correlates directly with the amount of FITC-labeled peptide within the. - cell: a higher value of. Relative MFI correlates with a larger amount of intracellular peptides labeled with FITC. The peptides in Table 23 were evaluated at concentrations of 0.63 μ ?, 2.5 μ? and 10 μ ?; PN768 was tested at 2 μ ?, 10 μ? and 50 μ ?. The truncated and total length forms of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides were exposed to the cells the day before transfection. The peptides labeled with FITC were diluted in Opti-MEM® medium (Invitrogen) for approximately 5 minutes at room temperature and then added to the cells. The cells were transfected for 3 hours and washed with PBS, treated with trypsin and then analyzed by flow cytometry. The cell viability was determined as in the above. Cellular uptake was distinguished from the membrane insert using trypan blue to fill all fluorescence on the surface of the cell membrane. For the analysis of cellular absorption, the peptide Total length PN73 labeled with FITC (PN690) achieved almost 100% cellular uptake at all concentrations tested (10 μ results shown in the column of Table 24 entitled "% Peptide Cell Absorption"). The remaining truncated forms of PN73, at a concentration of 10 μ except for PN768 which required .50 μ ?, achieved a percentage cellular uptake (values in parentheses) comparable to; that of PN690 indicating that N-terminal residues of PN73 are not required for the ability of the peptide to enter the cells. The five C-terminal residues of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides, identified as PN768, are sufficient for cellular uptake of the peptide. However, as a note and not shown in Table 24, the truncated forms of PN73 to 0.63 μ? showed, a decrease in cell absorption activity proportional to the peptide length. In other words, the general observation of the peptides tested at a concentration of 0.63 μ? is that, as the length of the PN73 peptide decreased, its cellular absorption activity decreased indicating that the activity of cellular absorption depends on the dose. These results showed that all truncated forms of the polypeptide that improves the delivery of exemplary polynucleotides retain the ability to be introduced to a high percentage of the cells in culture. Although the cellular uptake analysis showed that the truncated forms of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides had comparable cell absorption activities, the MFI measurements indicated that the truncated forms of the PN73 peptide were distinguishable based on the average amount of the FITC-labeled peptide that was introduced into the cells. As shown in the column entitled "FITC Peptide MIF" of Table 24, FITC-labeled full length PN73 peptide (PN690) showed a dose-dependent increase in MFI with 10 μ? achieving the highest MFI to approximately 125 units. The PN661, PN685 and PN660 had MFI levels comparable to those of full length PN73 (PN690). However, PN735 and PN655 had reduced MFI levels of approximately 80 MFI units and 60 MFI units, respectively. And, a significant decrease in the detection capacity of MFI was observed with PN654, PN708, PN653, PN652, PN651 and PN768 indicating that the efficient uptake of the peptide by the cells is inhibited by suppressing the 18 residues of the N-terminus of PN73. These results showed that all truncated forms of PN73 are capable of introducing a high percentage of cells in culture and, specifically, that the first 18 N-terminal residues of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides are essential for the efficient absorption of peptide by the cells in culture. These results showed that the truncated derivative of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides (PN661; PN685; PN660; PN735 and PN655) can be efficiently introduced into the cells in culture. In summary, the cellular uptake data of the peptide show that the five C-terminal residues of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides are sufficient for introduction to the cell indicating that the membrane / fusogenic binding domain of the peptide is localized in terminal C. However, the MFI data of the FITC peptide show that the removal of the eighteen N-terminal residues of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides limits the efficiency at which the peptide is introduced into the cells indicating that the N-terminus of the peptide is necessary for the efficient absorption characteristic of the membrane / fusogenic binding domain. Nucleotide Binding Domain of the Polypeptide that improves the supply of polynucleotides Exemplary: The ability of each peptide in the deletion series to complex and deliver the siRNA within primary MTF cells was measured by cell absorption analysis and by MFI. The nucleotide binding domain (s) of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides was characterized (characterized) by comparing the relative amount of cellular uptake of the siRNA of the different peptides in the deletion series. The peptides showed a high percentage of cellular absorption of the siRNA (40% or more), which correlates with the presence of a nucleotide binding region. A low percentage of cellular uptake of the siRNA (below 30%) reflected the absence of the nucleotide binding domain (s). To test the full length and truncated forms of the polypeptide that enhances the exemplary PN73 polynucleotide delivery, MTF cells were treated with siRNA or peptides conjugated to Cy5 or FAM as described above. After washing, the cells were treated with trypsin, and then analyzed by cytometry of flow. The intracellular uptake of the siRNA was measured by the intensity of the intracellular Cy5 fluorescence; Trypan blue was used to fill all fluorescence on the surface of the cell membrane. The absorption relative to the total number of cells was expressed. Table 24 shows the percentage results of the cellular uptake of siRNA of the peptide with 0.5 μ? of siRNA conjugated to Cy5. The peptide not labeled with FITC PN73 (PN643) achieved almost 100% absorption of siRNA at a concentration of 10 μ? (data not revealed) . However, when the PN73 peptide was labeled with the FITC label (PN690), its maximum cellular absorption activity, which was observed with 2.5 μ ?, was reduced to approximately 60% indicating that the addition of the FITC label interferes with the cell absorption function of the peptide. Therefore, the cellular uptake activity observed for each peptide in this analysis may not reflect the true cellular uptake activity of the siRNA. However, a slight decrease in the cellular uptake activity of the siRNA was observed by removing the three most extreme N-terminal residues of the peptide PN73 (PN690) as represented by PN661. Similarly, both PN660 and PN708 had moderate decreases in the cellular uptake activity of the siRNA compared to the full-length PN73 (PN690). These data show that the polypeptides of Enhancement of polynucleotide delivery of the invention including PN661, PN660 and PN708, binds to nucleic acids. In contrast, a reduction in siRNA uptake activity was observed with PN685, PN735, PN655 and PN654. No significant siRNA uptake activity was observed with PN653, PN652 or PN651, suggesting that the 12 terminal amino acids "C of the polypeptide enhancing the exemplary polynucleotide delivery (residues 37-48 of the H2B protein) do not contain a nucleotide binding domain Additionally, since the PN661, PN660 and PN708 peptides do not include three deletions of the full-length peptide residue (PN690) they still retain the binding activity of the inferred siRNA. nucleotide binding present, within the N-terminus of the polypeptide that enhances the delivery of exemplary polynucleotides to bind nucleic acids is sensitive to the presence of specific residues within the N-terminus. In general, these data show that the cell absorption activity of the Arnsi of the PN73 deletion series indicates that PN708 (residues 34-48) represents the minimum C-terminal fragment of the peptide do PN73 that is required for the cellular absorption activity of the Arnsi. This is consistent with the nucleotide binding domain of the polypeptide that enhances the delivery of exemplary PN73 polynucleotide is located in the first 24 residues of terminal N. The PN73 peptide deletion series was further characterized by its ability to transfect siRNA within cells by MFI, which determined the relative average amount of siRNA conjugated to Cy5 that was introduced into the cells. The delivery of the siRNA conjugated to Cy5 by the PN73 peptide labeled with FITC (PN690) achieved an MFI of approximately 50 relative units. The peptides PN735, PN655 ,. PN654 and PN708 of the PN73 suppression series showed a reduced MFI, which varies from approximately 34 units to '44 units. The PN661, PN685 and PN660 of the PN73 deletion series had MFI levels that were slightly higher than those of the full-length PN73 peptide (PN690). In contrast, PN654, PN653,, PN652 and PN651 had relatively little or no MFI (3-14 units) indicating that little or no siRNA conjugated to Cy5 was introduced into the cells after transfection with these peptides. The low MFI values observed with PN653, PN652 and PN651 correlate with the cellular uptake data of the siRNA that showed that PN654, PN653, PN652 and PN651 do not complex or facilitate the introduction of the siRNA within the cells. Microscopic fluorescence visualization was used to compare the cellular localization of Cy5-labeled siRNAs complexed with the polypeptide that improves the supply of PN73 polynucleotides with that of the siRNAs transfected with lipofectamine (Invitrogen). The location of the siRNA supplied with lipofectamine is characterized by a more punctuated staining, indicative of possible endosomal localization, while the PN73 exhibits a more uniform perinuclear staining. - The siRNA located in endosomes is not accessible to the RISC complex in the cytoplasm, unable to silence the target gene expression. In comparison, the uniform cytoplasmic distribution of the siRNA observed with the PN73-mediated delivery is a prerequisite for accessing the RISC complex, and for reducing target gene expression. The results show that the polynucleotide supply enhancement polypeptides of the invention substantially improve the. provision of siRNA over cationic lipids, and substantially improve the silencing of the gene. objective (deactivation). These data show that the ability of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides to improve the delivery of the siRNA with high efficiency within the cells depends on the 24 most extreme N-terminal residues of the peptide. These data show that shorter derivatives of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides (PN661; PN685; PN660; PN735; PN655; and PN708) can be complexed with and efficiently deliver the siRNA within the cells. Analysis of the Truncated Forms of the Polynucleotide Supply Enhancement Polypeptides PN360 and PN361: The structural activity activity relationships of the C-terminal and N-terminal regions of the PN73 were shown characterizing PN360 (C-terminal) ~ and PN361 (N-terminal) in a analysis of cellular absorption of siRNA carried out as described above. Table 24 shows that the deleted part of the N-terminus of PN73 (see PN361) reduced the cellular uptake activity of the siRNA by 50%; the removal of terminal C residues (see PN360) inhibits all cellular absorption activity of Arnsi. These data show that the C-terminal domain of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides is required for the cell-absorbing activity of the nucleotide of the peptide. Peptide-siRNA Conjugates Improved Covalently Linked Charge Delivery Within Cells: The cellular uptake activity of the peptide and the MFI measurements of the truncated forms of the polypeptide that enhances the exemplary polynucleotide delivery indicate that these peptides can function as delivery vehicles for a variety of molecular charges. This includes the covalent linkage of the desired effector molecule, including nucleic acids and peptides, to full length PN73 or its derivatives. The restricted delivery of the effector molecule to a particular cell type and / or organelle within the cells can be achieved by modifying the delivery peptide with a specific residue (lipid, peptide and / or sugar group). Analysis of deletion of the polypeptide that enhances the exemplary polynucleotide delivery suggests that the N-terminus is critical for the ability of the peptide to bind and deliver nucleotides (e.g., siRNA) within the cells. However, even in the absence of 'N' terminal residues, these nucleotide non-binding and severely compromised nucleotide binding peptides retain their membrane and fusogenic binding domains (eg, PN361; PN735; PN655; PN654; PN653; PN652 and PN651 and derivatives thereof). In light of the inability of the peptides to bind nucleotides, these peptides can still be used for ANSI delivery purposes within cells by covalently linking the ANsi to the membrane binding domains of the peptide and / or fusogenic. As a consequence, the convalent link of the ANsi remedies the inability of the peptide to bind nucleotides and allows the efficient delivery mediated by the peptide of the ANsi within the cells. In addition, the ANsi / peptide conjugates do not need to be limited to the selected truncated forms of the polypeptide that enhances the exemplary polynucleotide delivery, but may also include the full length PN73 and derivatives thereof. The following is a non-limiting example of the methods used to generate a covalent linkage of ANsi / peptide. The molecules of both the peptide and siRNA must be functionalized with specific residues to allow a covalent linkage between them. For the peptide, the N-terminal is functionalized, for example, with 3-maleimidopropionic acid. However, it is recognized that they also work. other functional groups such as bromine or iodoacetyl residues. For the RNA molecule the 5 'end of the sense chain or the end. 3 'of the chain of. antisense, is functionalized with for example, a 1-0-dimethoxytritylyl hexyl disulphide bond according to the following synthetic method. The ANsi modified at 5 'will be reduced to; free thiol group with 0.393 mg / 3 eq.) of tris (2-carboxyethyl) phosphine (TCEP) in 0.3 ml of 0.1 M of triethylamine acetate buffer (TEAA) (pH 7.0) at room temperature for 3 hours. The reduced oligonucleotide will be purified by RP HPLC on a 4.6 x 50 mm XTerra®MS Ci8 column using a linear gradient of 0 to 30% CH3CN in 0.1 M of TEAA buffer, pH 7, within 20 minutes (tr = 5.931 min. .).

Such conjugates were prepared as described above. The purified reduced ANsi (1361 mg, 0.19085 ymol) was dissolved in 0.2 ml of 0.1 TEAA buffer, pH = 7, and then the peptide with the maleimido residue bound to the N-terminus of the peptide (0.79 mg, 1.5 eq. ) and was added to the ANsi solution. "After the addition of the peptide, a precipitate was dissolved by the addition of 150 μ? Of 75% TEAA CH 3 CN / 0.1 M After stirring overnight at room temperature, the resulting conjugate was purified by RP HPLC on a column XTerra®MS Ci8 of 4.6 x 50 mm using a linear gradient of 0 to 30% CH3CN in 0.1 M of TEAA buffer, pH 7, within 20 minutes and 100% C within the next 5 minutes (tr = 21.007 min. The amount of the conjugate was determined by spectrometry based on the molar absorption coefficient calculated at λ = 260 mm The mass spectrometric analysis MALDI showed a peak observed for the conjugate (10 585.3 amu) equaling the calculated mass. sense and the antisense complementary strand of the peptide conjugate was hybridized in 50 mM potassium acetate, 1 mM magnesium acetate and 15 mM HEPES, pH 7.4 heating at 90 ° C for 2 minutes followed by incubation at 37 ° C for 1 hour. The formation of the double-stranded RNA conjugate was confirmed by undenatured polyacrylamide gel electrophoresis (15%) followed by stained with ethidium bromide. The results of the cell absorption activity are shown in the following Table 25. Table 25: Percentage of Cells that Absorb the siRNA by Peptide-siRNA Conjugates The results in the. Table 25 shows that the PN651-siRNA conjugate improves the absorption of the siRNA within the cells. Next, the MFI was measured. The results are presented in Table 26. Table 26: Relative Quantity (MFI) of Cy5-siRNA Supplied by Peptide-siRNA Conjugates The MFI results shown in Table 26 are consistent with the percent siRNA absorption data shown in Table 25. These data show that. the PN651-siRNA conjugate improved the absorption of siRNA within the cells. Target Gene Deactivation Activity of the Selected Truncated Forms of the Polypeptide that Enhances the Delivery of Polynucleotides Exemplary: Effective deactivation of the target gene expression was demonstrated by siRNA / polypeptide complexes that enhance the polynucleotide delivery of the invention. Specifically, the ability of the siRNA / polypeptide complexes to improve the delivery of polynucleotides to modulate the gene expression of human tumor necrosis factor-a (TNF-OI h) was evaluated. The significance of the direction of the h-TNF-a gene is that it is involved in mediating the occurrence or progress of rheumatoid arthritis (RA) when; over-expresses in human subjects or other mammals. Human monocytes were used as a model system to determine the effect of siRNA / polypeptide complexes that improves the delivery of polynucleotides on the genetic expression of hTNF-a. Qneg represents a random Arnsi sequence and works as the negative control. The observed Qneg deactivation activity is normalize at 100% (gene expression levels of 100%) and the deactivation activity of each of the following siRNAs, A19S21, 21 / 2.1 and LC20 was presented as a relative percentage of the negative control. A19S21, 21/21 and LC20 are the. ARNsis that are directed to hTNF-a mRNA. The PN643 polynucleotide PN643 (PN73 full-length minus a C-terminal), PN690 (full-length PN73 with a FITC at the C-terminal) and truncated PN73 from the deletion, polynucleotide PN643 polypeptides PN660, PN735, PN654 and PN708 form complejoron with the A19S21, 21/21 and LC20 siRNAs to determine their "effect on the ability of each siRNA to reduce the levels of genetic expression of hTNF-a in human monocytes. As follows: In a 9.6-well flat-bottom plate, human monocytes were seeded at 100K / well / 100 μl in OptiMEM medium (Invitrogen). Exemplary polynucleotide delivery enhancement polypeptides were mixed with 20 nM SiRNA at a molar ratio of 1 to 5 in OptiMEM medium at room temperature for 5 minutes At the end of the incubation, PBS was added to the mixture (final 3%), and 50 μl of the mixture was added to the cells. The cells were incubated at 3 7 ° C for 3 hours. After incubation, the cells were transferred to a V-bottom plate and granulated at 1500 rpm for 5 minutes. The cells were resuspended in growth medium (IMDM with glutamine, non-essential amino acid and peri-strep). After an overnight incubation, the monocytes were stimulated by applying LPS (Sigma) at 1 ng / ml for 3 hours to increase the expression of TNF-α expression levels. After induction by LPS, the cells were harvested as above for quantification of mRNA, and the supernatant was saved for protein quantification if desired. For the measurement of mRNA, branched DNA technology from Genospectra was used according to the manufacturer's specification. To quantify the level of mRNA in the cells, the mRNA of both the guardian gene (cypB) and the target gene (TNF-a) was measured and the reading, for TNF-a was normalized with cypB to obtain a unit of relative luminescence . The deactivation activity of the full-length and truncated forms of the polypeptide that enhances the delivery of exemplary PN73 polynucleotides is summarized in Table 24. A "+" in the "Deactivation Activity" column indicates that the peptide / SiRNA had an inactivation activity of 80% of the negative control Qneg siRNA (20% reduction in mRNA levels compared to the negative Qneg control). A "+/-" indicates that the peptide / siRNA complex had a deactivation activity of approximately 90% of the control siRNA negative Qneg (10% reduction in mRNA levels compared to the negative control of Qneg). Finally, a "-" indicates that the peptide / siRNA complex did not have a significant deactivation activity compared to the negative control of Qneg. In general, "PN643 (total length PN73 not labeled with FITC) and PN690 (full length PN73 labeled with FITC) had equivalent siRNA deactivation activities for all siRNAs tested as indicated by" + "in the column of "Deactivation Activity" (results shown in Table 24) Additionally, PN660 had siRNA deactivation activities for all tested siRNAs that were comparable to PN643 and PN690 indicating that the removal of the 9 N-terminal residues Peptide ends PN73 did not affect the RNAsis-mediated deactivation activity of TNF-α mRNA The PN654 showed moderate deactivation activity for both A19S21 and 21/21 siRNA but not for the: siRNA LC20 (deactivation activity shown by "+" in the deactivation activity column.) However, siRNAs complexed with either PN708 or PN735 did not result in an observable deactivation activity for none of the ARNsis. These results showed that the truncated forms of the polypeptide that improves the polynucleotide supply Exemplary PN73, specifically PN660 and PN654, do not interfere with the ability of siRNAs to lower mRNA levels of a target gene and offer a novel method for improving the delivery of therapeutic siRNAs for the treatment of human diseases such as RA. EXAMPLE 19 Characterization of the Polypeptide that Enhances the Supply of · PN708 Polynucleotides The present example further explores the cellular uptake activity of the siRNA, the MFI measurements and the deactivation activity of the siRNAs complexed with the PN708 peptide of the available peptides listed in the PN73 suppression series (refer to Table 23 in Example 17). As described above, the cell absorption analysis determines the number of cells receiving conjugated siRNA, with Cy5 upon complexing with a peptide. . The cellular uptake of Arnsi was evaluated by flow citometry. Absorption was expressed as a calculated percentage by dividing the number of cells containing siRNA conjugated with Cy5 between the total number of transfected and non-transfected cells in culture. The mean fluorescence intensity (MFI) was measured by flow cytometry and the amount of siRNA conjugated with Cy5 found within the cells was determined. The value of MFI correlates directly with the amount of siRNA conjugated with Cy5 within the cell, therefore, a higher value of MFI indicates a larger number of Cy5-conjugated siRNAs within the cells .. In the present example, a larger range of concentrations of peptide compared to the previous example to determine the efficiency of the cellular uptake activity of siRNA and the MIF measurements. In addition, cell viability was evaluated. In the present example, exemplary polynucleotide delivery enhancement polypeptides PN643 (full-length PN73 minus one C-terminal tag), PN690 (full-length PN73 with a FITC tag at C-terminal) and PN708 ( 15 mer derived by the deletion of the 21 N-terminal residues of PN73). at 5 μ ?, 10 μ ?, 20 μ? and 40 μ ?. The PN643 and the PN690 at 2.5 μ were also tested. and the PN690 was further tested at 1.25 μ ?. The PN643 and the PN708 were also tested both at 80 μ ?. As shown in Table 27 below, the PN73 peptide not labeled with FITC (PN643) achieved almost 100% absorption of the siRNA at a concentration of 10 μ ?. However, when the peptide PN73 was labeled with the FITC mark (PN690), its maximum cell absorption activity was reduced to approximately 70%. PN708 showed a dose-dependent increase in the cellular uptake activity of the siRNA. The PN708 achieved a cellular absorption activity maximum of the siRNA from 95% to 80 μ ?. For full-length PN73 peptides, cell viability decreased as the concentration of the peptide was increased. In contrast, cells incubated with the PN708 peptide maintained more than 90% cell viability in the presence of all concentrations tested. The MFI measurements of Cy5 further showed that the PN708 truncated peptide practically doubled the amount of Cy5 siRNA delivered within the. cells compared to the full length PN73 peptide (PN690). Table 27: Summary of the Supply Improvement Characteristics of the PN708 siRNA These results showed that the polypeptide that enhances the PN768 truncated polynucleotide delivery exemplary (residues H2B 34-38) has the ability to improve the delivery of the siRNA with high efficiency within cells without adversely affecting cell viability. The polypeptide that improves the delivery of exemplary PN708 truncated polynucleotides was further characterized by determining whether affectation in the reduction of target gene expression mediated by siRNA. In this example section, the FITC tag at the C terminal of the PN708 peptide was removed prior to assessing its ability to improve the reduction of target gene expression by complexing with an siRNA. In the absence of the FITC tag, the polypeptide that enhances the exemplary truncated polynucleotide delivery was designated PN766 (refer to Table 23 in Example 17). The ability of the siRNA / peptide complexes to modulate the gene expression of human tumor necrosis factor-a (hTNF-cx) was evaluated. In the present example, the sequence, of random siRNA, Qneg, served as a negative control and they used the LC20 and LC17 siRNAs to direct hTNF-a mRNA in human monocytes. The molar ratios of siRNA to peptide tested were 1: 5; 1:10; 1:25; 1:50; 1:75 and 1: 100. Both LC20 and LC17 were used at a concentration of 20 nM. The deactivation results show that the siRNA / peptide complexes of both LC20 / PN766 and LC17 / PN766 at 1: 5; 1:10; and 1: 25 -reduced hTNF-a mRNA levels to approximately 70% -80% of the negative control of Qneg siRNA (ie, 20% -30% reduction in mRNA levels compared to the negative control of Qneg). The siRNA / peptide ratios of 1:50; 1:75 and 1: 100 had no significant effects on hTNF-a mRNA levels compared to the Qneg control. No cytotoxicity effects were observed with human monocytes in the presence of the peptide PN766. These data showed that the polypeptide that enhances the PN766 exemplary polynucleotide delivery by complexing with siRNA significantly reduces mRNA levels of the target gene indicating that PN766 is an ideal siRNA delivery peptide for therapeutic siRNAs in the treatment of RA in subjects mammals EXAMPLE 20 Substitutions and Suppressions of Amino Acid within the Polypeptide that improves the supply of polynucleotides Exemplary Do not Affect the Cell Absorption Activity of the Peptide Mediated siRNA The present example demonstrates that the exemplary mutant polynucleotide delivery enhancement polypeptides listed in Table 28 below, generated by substitution and / or deletion of the residue, did not affect the activity of cellular uptake of the siRNA or MFI measurements compared to the polypeptide that enhances the PN73 exemplary unmodified polynucleotide delivery. Table 28 below represents the substitutions of the residue effected within the polypeptide that enhances the delivery of exemplary PN73 polynucleotides. The gray-enhanced amino acids represent the substituted and / or deleted unmodified residues. The gray enhancement allows easy identification and comparison of the unmodified residues within the PN73 peptide with the substituted and / or deleted residues within the exemplary mutant polynucleotide delivery enhancement polypeptides PN644, PN645, PN646, PN647 and PN729. The substituted residues within the exemplary mutant polynucleotide delivery enhancing polypeptides are in bold and underlined. Also, the symbol "?" in bold and underlined represents a residue deleted within PN729.

The purpose of generating exemplary mutant polynucleotide delivery enhancing polypeptides by substitution or deletion of the residue was to evaluate the effect of these modifications on the cellular uptake activity of the siRNA and the efficiency at which the siRNAs are introduced into the cells. Table 28: PN73 Waste Substitution and Suppression Series X = represents a substituted amino acid; * = deleted amino acid; Specific substitutions and / or deletions of the residue within the polypeptide that enhances the delivery of exemplary PN73 polynucleotides include the change or increase in the number of aromatic amino acids and / or the decrease in the number of negatively charged amino acids. The amino acids with aromatic functional groups (eg, phenylalanine, tyrosine, tryptophan and their derivatives) are typically found in the protein membrane expansion domains due to their relatively non-polar character (hydrophobic) and their ability to facilitate the penetration of the cell membrane of the protein. negatively charged amino acids repel the negatively charged phosphodiester structure of nucleic acids and thus impair the ability of a protein to bind nucleic acids. Therefore, the reasons for amino acid substitutions Aromatics within the PN73 peptide include improving the cell penetration function of the peptide and / or the removal of the negatively charged amino acids to improve the nucleic acid binding of the peptide. The. Peptide nucleic acid binding capabilities can also be promoted simply by deleting the amino acids. negatively charged or substituting the negatively charged amino acid with a positively charged or neutral amino acid. The analysis of cellular uptake of the siRNA and the MFI measurements were carried out as previously described. The data is summarized in Table 29 below. Each peptide was tested at concentrations of 0.63 μ?, 1.25 μ ?, 2.5 μ? and 5 μ ?. The results show that despite the substitutions and / or deletions of the residue within the polypeptide that improves the delivery of exemplary PN73 polynucleotides, the activity of. Cellular uptake of the siRNA and MFI measurements of the mutant peptides remained equivalent to those of the unmodified PN73. These data indicate that substitutions and / or deletions of the residue did not affect the ability of the peptide to bind nucleic acids and. penetrate the cell membrane. In addition, cell viability was not affected by the presence of substituted and / or deleted residues within the polypeptide that enhances the exemplary PN73 polynucleotide delivery. Table 29: Summary of the Supply Characteristics of the siRNA Mediated by PN73 Mutant The results showed that modifications of the polypeptide that enhances the delivery of exemplary polynucleotides generated for example, by substitutions or deletions of amino acids or combinations thereof, provide the siRNA to a high percentage of cells with high efficiency. EXAMPLE 21 Cell Absorption Activity of siRNA mediated by the polypeptide that enhances polynucleotide delivery The present example illustrates the efficiency of the cellular uptake activity of the siRNA for the polynucleotide delivery enhancement polypeptides listed in Table 30 complexed with siRNA. Table 31 summarizes the data on the cellular uptake of the siRNA, the measurements of mean fluorescence intensity (MFI) and cell viability data for each of the polypeptides. The polypeptides that achieved a percentage of cellular absorption of the siRNA of 75% or greater are highlighted in gray in the "treatment" column. the cellular uptake of the percentage siRNA specific for each of these siRNA / peptide complexes is also highlighted in gray on the "% cell uptake of siRNA" column. Table 30: Supply Improvement Polypeptides Visualized by their Activity Cell Absorption of siRNA The analysis of. Cellular uptake of siRNA in the present example determines the number of cells that received siRNA LC20 conjugated to Cy5 in the presence of the peptide. LC20 is an oligo used to direct the mRNA of mRNA of tumor necrosis factor-alpha (hTNF-a). The absorption of the siRNA by the cells was evaluated by flow cytometry. Absorption was expressed as a percentage calculated by dividing the number of cells containing siRNA conjugated with Cy5 between the total number of cells transfected and non-transfected in culture. The mean fluorescence intensity (MFI) was measured by flow cytometry and the amount of siRNA conjugated with Cy5 found within the cells was determined. The value of MFI is. correlates directly with the amount of siRNA conjugated with Cy5 within the cell, therefore, a higher value of MFI indicates a greater number of siRNAs conjugated with Cy5 within the cells. The following protocol was used to test the polynucleotide supply enhancement polypeptides listed in Table 30. Approximately 80,000 mouse tail fibroblast (MTF) cells were plated per well in 24-well plates the day before the transfection in complete medium. Each delivery peptide, except the positive control, was tested at concentrations of 0.63 μ ?, 2.5 μ ?, 10 μ? and 40 μ? in the presence of 0.5 μ? of the conjugated siRNA with Cy5. For, the siRNA / peptide complexes, the siRNA conjugated with Cy'5 and the peptide were diluted separately in OptiME ® medium (Invitrogen) at two times the final concentration. Equal volumes of siRNA and peptide were mixed and allowed to complex for five minutes at room temperature. The siRNA / peptide complexes were added to cells previously washed with phosphate buffered saline (PBS). The cells were transfected for three hours at 37 ° C, 5% C02.The cells were washed with PBS, treated with trypsin and then analyzed by flow cytometry.The cellular uptake of the siRNA was measured by the intensity of the cells. Fluorescence of intracellular Cy5 cell viability was determined using propidium iodide uptake or staining with Annexin V-PE (BS Biosciences) In order to differentiate the cellular uptake of the membrane insert from the labeled siRNA (or labeled peptide with fluorescence)Trypan blue was used to fill all fluorescence on the surface of the cell membrane. Trypan blue (Sigma) was added to the cells at a final concentration of 0.04% and re-introduced into the flow cytometer to evaluate if there was any change in fluorescence intensity which would indicate localized fluorescence in the cell membrane.

Table 31: Screen Data of the siRNA Delivery Mediated by the Polypeptide (NT = not tested) As shown in the column titled "% cell uptake of the siRNA" of Table 31, the "no treatment" negative control showed no cellular uptake of siRNA while the positive control peptide achieved a percentage siRNA cellular uptake activity of 95%. The LC20 siRNA conjugated to Cy5 complexed with polynucleotide delivery enhancement polypeptides PN680; PN681; PN709; PN760; PN759 or PN682 achieved an absorption activity percentage siRNA cell that exceeded 75% or higher. The PN694 and PN714 polynucleotide supply enhancement polypeptides exhibited a moderate siRNA cellular uptake activity of 54% and 43% respectively. In contrast, the polynucleotide delivery enhancement polypeptides PN665 and PN734 did not demonstrate any significant siRNA uptake activity (less than 5%). The polynucleotide delivery enhancement polypeptides were further characterized by their ability to transfect siRNA within the cells by analyzing the mean fluorescence intensity (MFI). Although the cellular absorption analysis determined the percentage of cells containing the siRNA conjugated to Cy5, the measurement of MFI determined the relative average amount of siRNA conjugated to Cy5 that was introduced into the cells. As shown in the column entitled "MFI of the Cy5 siRNA" of Table 31, delivery of the siRNA conjugated to Cy5 by the positive control peptide PN643 achieved an MFI of approximately seven units. As expected, the negative control "without treatment" did not have a calculable MFI. The polypeptide that improves the PN665 polynucleotide delivery was not tested by MFI. PN743, PN694 and PN714 had significantly lower MFI measurements than the positive control. The PN680, PN709 and PN682 polynucleotide supply enhancement polypeptides exhibited comparable MFI measurements with those of the positive control PN643 while the PN681 had a MFI double that of the positive control. Surprisingly, the PN760 and PN759 polynucleotide supply enhancement polypeptides had MFI measurements that were approximately 13 times and 6 times higher, respectively, than those of the positive control. These data show that PN680 polynucleotide supply enhancement polypeptides; PN681; PN709; PN760; PN759 and PN682 when complexed with siRNA efficiently deliver the siRNA within the cells. EXAMPLE 22 Activity of Deactivation of Gene Expression of Transfected RNAsis Within Cells with Polynucleotide Supply Improvement Polypeptides The present example demonstrates that siRNA complexed with polynucleotide delivery enhancement polypeptides effectively inactivates mRNA expression of the target gene of the polynucleotide. SiRNA. : Specifically, the ability of the siRNA / peptide complexes to modulate the expression of the human tumor necrosis factor (hTNF-a) was evaluated. The significance of the direction of the hTNF-a gene is that it is involved in mediating the occurrence or progress of rheumatoid arthritis (RA) when it is over-expressed in human subjects or other mammals. Human monocytes were used as a system of model to determine the effect of siRNA / peptide complexes on the genetic expression of hTNF-a. Qneg represents a random siRNA sequence. and that works like the negative control. The observed Qneg deactivation activity is normalized to 100% (gene expression levels of 100%) and the deactivation activity of each of the following siRNAs, A19S21 MD8, 21/21 MD8 and LC20 was presented as a relative percentage of the negative control. A19S21 MD8, 21/21 MD8 and LC20 are the siRNAs that target hTNF-a mRNA. The 'polypeptide that improves the supply of polynucleotides PN602 represented an acetylated form of the positive control used in the previous Examples and is used herein as a positive control both for the effective delivery of the siRNA in human monocytes and for the permissive activity of deactivation of hTNF-a mRNA levels mediated by SiRNA .. The PN680 and PN681 polynucleotide supply enhancement polypeptides form complejoron with the siRNAs listed above to determine their effect on the ability of each siRNA to reduce the levels of genetic expression of hTNF-a in human monocytes. The deactivation activity of all three polynucleotide supply enhancement polypeptides, is summarized below in Table 32. A "+" in the "Deactivation Activity" column indicates that the peptide / siRNA complex had a deactivation activity of 80% of the negative control Qneg siRNA (20% reduction in mRNA levels compared to the negative Qneg control). A "+/-" indicates that the peptide / siRNA complex had a deactivation activity of approximately 90% of the Qneg negative control siRNA (10% reduction in mRNA levels compared to the negative control of Qneg). Finally, a "-" indicates that the peptide / siRNA complex did not have one. Significant deactivation activity compared to the negative control of Qneg. Transfections in human monocytes for the present example were carried out according to previously described protocols. For the measurement of the mRNA, branched DNA technology from Genospectra (CA) was used according to the manufacturer's specification. · To quantify the level of mRNA in the cells, both the guardian gene (cypB) and the mRNA were measured. target gene (TNF-), and the reading for TNF-cx was normalized with cypB to obtain a relative luminescence unit. Table 32: RNAi Deactivation Activity for Complex RNAse 'with Polynucleotide Supply Improvement Polypeptides The results shown in Table 32 indicate that all three siRNAs complexed with the polypeptide that improves the delivery of positive control PN602 polynucleotides at ratios of 1: 5 and 1:10, moderately lowered the levels of genetic expression of hTNF- a compared to the negative control Qneg complexed with the same polypeptide. However, the same siRNAs complexed with the polypeptide that improves, the supply of. polynucleotides PN681 at 1: 5 and 1:10 showed little or no deactivation activity in relation to the Qneg complex of siRNA / PN681 negative control. In contrast, the polypeptide that enhances the PN680 polynucleotide delivery complexed with any of the hTNF-a-specific siRNAs at a ratio of 1: 5 exhibited significant deactivation activity of the hTNF-a mRNA relative to the control complex. Qneg / PN680. In addition, the LC20 / PN680 complex to a relationship of 1:10 also demonstrated significant deactivation activity compared to the control complex of Qneg / PN680. These data show that the polypeptide that improves the PN680 polynucleotide delivery delivers the siRNAs within the cells and allows for the effective silencing of the siRNA mediated gene. Although the above invention has been described in detail by way of example for purposes of clarity and understanding, it will be apparent to the person skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims which are presented by way of illustration and not limitation. . In this context, several have been cited. publications and other references within the above description by economy of description. Each of these references is incorporated herein by reference in its entirety for all purposes. However, it is noted that the various publications discussed herein are incorporated only for their description prior to the date of filing of the present application, and that the inventors reserve the right to antedate such description by virtue of the prior invention.

Claims (37)

1. CLAIMS 1. A composition comprising a polypeptide that improves the supply of polynucleotides and. a double-stranded ribonucleic acid (dsRNA), wherein the polypeptide that improves the polynucleotide delivery is amphipathic and comprises nucleic acid binding properties. The composition of claim 1, wherein the polypeptide that enhances the polynucleotide delivery comprises from about 5 to about 40 amino acids, and has all or part of a sequence selected from the group consisting of Poly (Lys, Tryp) 4: 1 MW 2.0,000-50,000, Poly (Orn, Trp) 4: 1 20,000-50,000, Melitin, Histone Hl, Histone H3 and Histone H4, SEQ ID Nos. 27 to 31, 35 to 42, 45, 47, 50 a 59, 62, 63, 67., 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178 and 180 to 186. 3. The composition of claim 1, wherein, the composition causes the absorption of dsRNA in an animal cell. 4. The composition of claim 3, wherein the animal cell is a mammalian cell. The composition of claim 1, wherein the composition is administered to an animal. 6. The composition of claim 5, wherein the animal is a mammal. 7. The composition of claim 1, in wherein the N-terminus of the polypeptide that improves the polynucleotide delivery is acetylated. The composition of claim 1, wherein the N-terminus of the polypeptide that enhances the delivery of polynucleotides is pegylated. The composition of claim 1, wherein the dsRNA is a small interfering ribonucleic acid (siRNA) consisting of a sequence of about 10 to about 40 base pairs that is complementary to a portion of an alpha-factor gene of tumor necrosis (TNF-). The composition of claim 1, wherein the dsRNA is an siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID Nos. 109 to 163 and 187. 11. The composition of claim 1, wherein the polypeptide enhances the delivery of. polynucleotides are mixed, complexed or conjugated to the dsRNA. 12. The composition of claim 1, wherein the polypeptide that enhances the polynucleotide delivery binds to the dsRNA. The composition of claim 1 further comprising a cationic lipid. 14. The composition of claim 13, wherein the cationic lipid is selected from the group consisting of N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride, 1,2-bis ( oleoyloxy) -3, 3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N trifluoroacetate -dimethyl-1-propanaminium, 1,3-dioleoyloxy-2- (6-carboxyespermyl) -propylamide, 5-carboxymethylglycine dioctadecylamide, tetramethyltetrapalmitoyl spermine, tetramethyltetraoleyl spermine, tetramethyltetralauryl spermine, tetramethyltetramiristyl spermine and tetramethyldioleyl spermine,. DOT A (N- [1- (2, 3-dioleoyloxy) propyl] -?,?,? - trimethyl ammonium chloride, DOTAP (1,2-bis (oleoyloxy) -3,3- (trimethylammonium) propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide), DDAB (dimethyl dioctadecyl ammonium bromide), cationic polyvalent lipids, lipoespermines, DOSPA (2,3-dioleyloxy-N- [trifluoro-acetate] 2-sperminecarboxamido) ethyl] -N, N-dimethyl-l-propanaminium), DOSPER (1,3-dioleoyloxy-2- (6-carboxy spermyl) -propyl-amide, di- and tetra-alkyl-tetra-methyl spermines , TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMS (tetramethyltetramiristyl spermine), TMDOS (tetramethyldioleyl spermine), DOGS (dioctadecyl-amidoglycyl spermine) (TRANSFECTA ®), cationic lipids combined with non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DphPE (difitanoylphosphatidylethanolamine) or cholesterol, a cationic lipid composition composed of a 3: 1 (w / w) mixture of DOSPA and DOPE, and a 1: 1 mix (weight / weight) of DOTMA and DOPE. 15. A method for causing the absorption of a double-stranded ribonucleic acid (dsRNA) in an animal cell, comprising incubating the animal cells with a mixture comprising a polypeptide that improves the supply of polynucleotides and the dsRNA, wherein the polypeptide is- improves the polynucleotide supply is amphipathic and comprises nucleic acid binding properties. 16. A method for modifying the expression of a target gene in an animal cell, comprising incubating the animal cell with a mixture comprising a polypeptide that enhances the polynucleotide delivery, wherein the polypeptide that enhances the polynucleotide delivery is amphipathic and comprises nucleic acid binding properties, and a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA is complementary to a region of the obj ective gene. 17. The method of claim 15 or 16, in where the animal cell is a mammalian cell. A method for changing a phenotype of an animal subject, comprising administering to the animal subject a mixture of a polypeptide that enhances the polynucleotide delivery, wherein the polypeptide that enhances the polynucleotide delivery is amphipathic and comprises acid binding properties nucleic acid, and a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA is complementary to a region of a target gene in the subject. 19. The method of claim 18, wherein the animal is a mammal. 20. The method of claim 15, 16 or 18, wherein the polypeptide "enhancing the polynucleotide delivery comprises from about. 5 to about 40 amino acids, and has all or part of a sequence selected from the group consisting of Poly (Lys, Tryp) 4: 1 MW 20,000-50,000, Poly (Orn, Trp) 4: 1 20,000-50,000, Melitin, Histone Hl, Histone H3 and Histone H4, SÉQ ID Nos. 27 to 31, 35 to 42, 45, 47, 50 to 59, 62, 63, 67, 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178 and 180 to 186. 21. The method of claim 15, 16 or 18, wherein the N-terminus of the polypeptide that improves the polynucleotide delivery is acetylated. 22. The method of claim 15, 16 or 18, wherein the N-terminus of the polypeptide that improves delivery of polynucleotides is pegylated. 23. The method of claim 15, 16 or 18, wherein the dsRNA. is a small interfering ribonucleic acid (siRNA) consisting of a sequence of about 10 to about 40 base pairs that is complementary to a portion of a tumor necrosis factor-alpha gene (TNF-α). The method of claim 15, 16 or 18, wherein the dsRNA is an siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID Nos. 109 to 163 and 187. 25. The method of claim 15, 16 or 18, wherein the polypeptide that enhances the polynucleotide delivery is mixed, complexed or conjugated to the dsRNA. .26. The method of claim 15, 16 or 18, wherein the polypeptide that improves the polynucleotide delivery binds to the dsRNA. 27. The method of claim 15, 16 or 18, further comprising a cationic lipid. The method of claim 27, wherein the cationic lipid is selected from the group consisting of N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride, 1-2. bis (oleoyloxy) -3, 3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, -dimethyl-l-propanaminium trifluoroacetate, , 3-dioleoyloxy-2- (6-carboxyespérilyl) -propylamide, 5-carboxymethylglycine dioctadecylamide, tetrámetiltetrapalmitoyl spermine, tetramethyltetrolyl spermine, tetramethyltetralauryl espérmita, tetramethyltetramiristyl spermine and tetramethyldioleyl spermine, DOT A (N- [1- (2, 3 -dioleoyloxy) propyl] -N, N, -trimethyl ammonium, DOTAP (1, 2-bis (oleoyloxy) -3, 3- (trimethylammonium) propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide), DDAB (bromide) of dimethyl dioctadecyl ammonium), cationic polyvalent lipids, lipoespermines, DOSPA ((2,3-dioleyloxy-N- [2-sperminecarboxamido) ethyl] -N, N-dimethyl-l-propanaminium trifluoro-acetate), DOSPER. (1,3-dioleoyloxy-2- (6-carboxy spermyl) -propyl-amide, di- and tetra-alkyl-tetra-methyl spermines, TMTPS (tetrámetiltetrapalmitoil espermina), TMTOS (tetramethyltetrolylethyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMS (tetramethyltetramiristyl spermine), TMDOS (tetramethyldioleyl spermine), DOGS (dioctadecyl-amidoglycyl spermine) (TRANSFECTAM®), cationic lipids combined with non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DphPE (difitanoylphosphatidylethanolamine) or cholesterol, a cationic lipid composition composed of a 3: 1 (w / w) mixture of DOSPA and DOPE, and a 1: 1 (w / w) mixture of DOTMA and DOPE. 29. The use of a mixture comprising a polypeptide that enhances the delivery of polynucleotides, wherein the polypeptide that enhances the delivery of polynucleotides is antipathetic and comprises binding properties to nucleic acid, and a double-stranded ribonucleic acid (dsRNA) for production of a medicament for the treatment of an inflammatory condition (s) associated with tumor necrosis factor-alpha (TNF-a) in an animal subject, wherein the drug is capable of reducing RNA levels of TNF-, thus preventing or reducing the occurrence or severity of one or more symptoms of the inflammatory condition (s) associated with TNF-a. 30. The 'use. of the mixture of claim 29, wherein the polypeptide that improves the polynucleotide delivery comprises from about 5 to about 40 amino acids, and has all or part of a sequence selected from the group consisting of 4: 1 Poly (Lys, Tryp). MW 20,000-50,000, Poly (Orn, Trp) 4: 1 20,000-50,000, elitin, Histone Hl, Histone H3 and Histone H4, SEQ ID Nos. 27 to 31, 35 to 42, 45, 47, 50 to 59, 62 , 63, 67, 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178 and 180 to 186. 31. The use of the mixture of claim 29, wherein the N-terminus of the polypeptide that improves the polynucleotide delivery is acetylated. 32. The use of the mixture of claim 29, wherein the N-terminus of the polypeptide that improves the polynucleotide delivery is pegylated. 33. The use of the mixture of claim 29, wherein the dsRNA is a small interfering ribonucleic acid (siRNA) consisting of a sequence of about 10 to about 40 base pairs that is complementary to a portion of the tumor necrosis factor-alpha (TNF-x). 34. The use of the mixture of claim 29, wherein the dsRNA is an siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID Nos 109 to 163 and 187 .. The use of the mixture of claim 29, wherein the polypeptide that improves the polynucleotide delivery is mixed, complexed or conjugated to the dsRNA. 36. The use of the mixture of claim 29, wherein the polypeptide that improves the polynucleotide delivery binds to the dsRNA. 37. The use of the mixture of claim 29, where the. animal subject is a mammal.