HK1123069A - Pharmaceutical compositions for delivery of ribonucleic acid to a cell - Google Patents

Description

Pharmaceutical compositions for delivering ribonucleic acid to cells

Technical Field

The present invention relates to methods and compositions for delivering ribonucleic acids into cells. More particularly, the invention relates to methods and agents for delivering double-stranded polynucleotides into cells to modify the expression of target genes, alter phenotypes such as a cellular disease state or potential.

Background

The delivery of nucleic acids into animal and plant cells has long been an important goal of molecular biological research and development. Recent advances in the fields of gene therapy, antisense therapy, and RNA interference (RNAi) therapy have created a need to develop more efficient methods for introducing nucleic acids into cells.

A wide variety of plasmids and other nucleic acid "vectors" have been developed for the delivery of large polynucleotide molecules into cells. Typically these vectors are combined with large DNA molecules containing the entire gene used to transform the target cell to express the gene for scientific or therapeutic purposes.

The process of artificial delivery of exogenous nucleic acids into cells is commonly referred to as transfection. Cells can be transfected with a number of techniques and materials to take up exogenous functional nucleic acids. The most commonly used transfection methods are calcium phosphate transfection and electroporation. Many other methods of transducing cells to deliver exogenous DNA or RNA molecules have been developed, including virus-mediated transduction, delivery of cationic lipids or liposomes, and many methods of targeting mechanical or biochemical membrane disruption/permeation (e.g., using detergents, microinjection, or ion guns).

RNA interference is a process of sequence-specific post-transcriptional gene silencing in cells initiated by a double-stranded (ds) polynucleotide, typically a dsRNA that is homologous in sequence to a portion of the target messenger RNA (mrna). The introduction of a suitable dsRNA into a cell results in the destruction of endogenous, homologous mRNAs (i.e., mRNAs that share substantial sequence identity with the introduced dsRNA). The dsRNA molecules are cleaved by so-called dicer RNase III family nucleases into small interfering RNAs (siRNAs) that are 19-23 nucleotides (nt) in length. The siRNAs are then incorporated into a multi-component nuclease called the RNA-induced silencing complex or "RISC". The RISC recognizes mRNA substrates through their homology to the siRNA and accomplishes silencing of gene expression through binding and disruption of the target mRNA.

RNA interference is a promising technology for the emerging modification of plant and animal specific gene expression and is therefore expected to provide a useful tool for the treatment of a wide variety of diseases and conditions treatable by modification of endogenous gene expression.

There remains a long-felt need in the art for better tools and methods for delivering siRNAs and other small inhibitory nucleic acids (siNAs) into cells, particularly in view of the fact that existing techniques for delivering nucleic acids to cells are limited by the low efficiency and/or high toxicity of the delivery agents. There is a need for methods and formulations for mediating the regulation of gene expression regulation in a manner that alters the phenotype or disease state of a target cell by delivering an effective amount, active and persistent state of siNAs to the selected cell, tissue or compartment using a non-toxic vehicle.

Summary of The Invention

One aspect of the invention is a composition comprising a polynucleotide delivery-enhancing polypeptide and a double-stranded ribonucleic acid (dsRNA), wherein the polynucleotide delivery-enhancing polypeptide is amphiphilic and comprises nucleic acid binding properties. In a related embodiment, the polynucleotide delivery-enhancing polypeptide comprises 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: 1MW20,000-50,000, poly (Orn, Trp) 4: 1MW20,000-50,000, melittin, histone H1, histone H3 and histone H4, SEQ ID NOS 27-31, 35-42, 45, 47, 50-59, 62, 63, 67, 68, 73, 74, 76, 78-87, 89-92, 94-108, 164-178 and 180-186.

In another embodiment, the composition results in uptake of the 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 polynucleotide delivery-enhancing polypeptide is acetylated. In a related embodiment, the N-terminus of the polynucleotide delivery-enhancing polypeptide 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 that is complementary to a portion of the gene of tumor necrosis factor-alpha (TNF-alpha). In a related embodiment, the dsRNA is encoded by a sequence selected from the group consisting of SEQ ID NOS: 109, 163, and 187 from about 10 to about 40 base pairs. In another embodiment, the polynucleotide delivery-enhancing polypeptide is mixed, complexed or conjugated to a dsRNA. In another embodiment, the polynucleotide delivery-enhancing polypeptide binds to the dsRNA. In 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 ] -N, N, N-trimethylammonium chloride, 1, 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane, 1, 2-ditetradecyloxypropyl-3-dimethylhydroxyethylammonium bromide, dioctadecyldimethylammonium bromide, 2, 3-dioleoyloxy-N- [2 (spermine carboxylic acid)Carboxamido) ethyl]N, N-dimethyl-1-propylammonium trifluoroacetate, 1, 3-dioleoyloxy-2- (6-carboxyspermine) -propionamide, 5-carboxysperminamine aminodiacetic acid dioctadecylamide, tetramethyltetrapalmitospermine, tetramethyltetraoleospermine, tetramethyltetralauryl spermine, tetramethyltetramyristyl spermine and tetramethyldioleospermine, DOTMA (N- [ 1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane), DMRIE (1, 2-bistetradecyloxypropyl-3-dimethylhydroxyethylammonium bromide), DDAB (dioctadecyldimethylammonium bromide), Polyvalent cation liposome, lipospermine, DOSPA (2, 3-dioleoyloxy-N- [2 (spermine carboxamide) ethyl)]-ammonium N, N-dimethyl-1-propyltrifluoroacetate), DOSPER (1, 3-dioleoyloxy-2- (6-carboxyspermine) -propionamide, di-and tetra-alkyl-tetra-methyl spermine), TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleoyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMS (tetramethyltetramyristyl spermine), TMDOS (tetramethyldioleoyl spermine) DOGS (dioctadecylamidoglycyl spermine (TRANSFECTAM)) Cationic lipid compositions consisting of cationic lipid bound to non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol, a 3: 1(w/w) mixture of DOSPA and DOPE, and a 1: 1(w/w) mixture of DOTMA and DOPE.

Another aspect of the invention is a method of causing uptake of a double-stranded ribonucleic acid (dsRNA) into an animal cell, comprising incubating the animal cell with a mixture comprising a polynucleotide delivery-enhancing polypeptide and the dsRNA, wherein the polynucleotide delivery-enhancing polypeptide is amphiphilic and comprises a nucleic acid binding property.

Another aspect of the invention is a method of modifying the expression of a target gene in an animal cell comprising incubating the animal cell with a mixture comprising a polynucleotide delivery-enhancing polypeptide and a dsRNA, wherein the polynucleotide delivery-enhancing polypeptide is amphipathic and comprises nucleic acid binding properties, wherein the dsRNA is complementary to a region of the target gene.

In related embodiments, in any of the above methods, the animal cell is a mammalian cell.

Another aspect of the invention is a method of altering a phenotype of an animal subject comprising administering to the animal subject a mixture of a polynucleotide delivery-enhancing polypeptide and a double-stranded ribonucleic acid (dsRNA), wherein the polynucleotide delivery-enhancing polypeptide is amphipathic and comprises nucleic acid binding properties, wherein the dsRNA is complementary to a target gene region of the subject. In related embodiments, the animal may be a mammal.

In another embodiment, the polynucleotide delivery-enhancing polypeptide in any of the above methods comprises 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: 1MW20,000-50,000, poly (Orn, Trp) 4: 1MW20,000-50,000, melittin, histone H1, histone H3 and histone H4, SEQ ID NOS 27-31, 35-42, 45, 47, 50-59, 62, 63, 67, 68, 73, 74, 76, 78-87, 89-92, 94-108, 164-178 and 180-186. In related embodiments, the N-terminus of the polynucleotide delivery-enhancing polypeptide in any of the above methods is acetylated. In another related embodiment, the N-terminus of the polynucleotide delivery-enhancing polypeptide in any of the above methods 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 that is complementary to a portion of a tumor necrosis factor-alpha (TNF- α) gene in any of the above methods. In related embodiments, the dsRNA described in any of the above methods is an siRNA consisting of about 10 to about 40 base pair sequence selected from the group consisting of SEQ ID NOS 109-163 and 187. In another embodiment, the polynucleotide delivery-enhancing polypeptide in any of the above methods is mixed, complexed or conjugated to a dsRNA. In another embodiment, the polynucleotide delivery-enhancing polypeptide binds to the dsRNA in any of the above methods. In another embodiment, any of the above methods further comprises a cationic lipid. In one phaseIn a related embodiment, the cationic lipid of any of the above methods 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-ditetradecyloxypropyl-3-dimethylhydroxyethylammonium bromide, dioctadecyldimethylammonium bromide, 2, 3-dioleoyloxy-N- [2 (spermine carboxamido) ethyl]ammonium-N, N-dimethyl-1-propyltrifluoroacetate, 1, 3-dioleoyl-2- (6-carboxyspermine) -propionamide, 5-carboxysperminamine aminoglycinate dioctadecylamide, tetramethyltetrapalmitospermine, tetramethyltetraoleospermine, tetramethyltetralauryl spermine, tetramethyltetramyristyl spermine and tetramethyldioleospermine, DOTMA (N- [ 1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane), DMRIE (1, 2-bistetradecyloxypropyl-3-dimethylhydroxyethylammonium bromide), DDAB (dioctadecyldimethylammonium bromide), Polyvalent cation liposome, lipospermine, DOSPA (2, 3-dioleoyloxy-N- [2 (spermine carboxamide) ethyl)]-ammonium N, N-dimethyl-1-propyltrifluoroacetate), DOSPER (1, 3-dioleoyloxy-2- (6-carboxyspermine) -propionamide, di-and tetra-alkyl-tetra-methyl spermine), TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleoyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMS (tetramethyltetramyristyl spermine), TMDOS (tetramethyldioleoyl spermine) DOGS (dioctadecylamidoglycyl spermine (TRANSFECTAM)) Cationic lipid compositions consisting of cationic lipid bound to non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol, a 3: 1(w/w) mixture of DOSPA and DOPE, and a 1: 1(w/w) mixture of DOTMA and DOPE.

Another aspect of the invention is the use of a mixture comprising a polynucleotide delivery-enhancing polypeptide and a double-stranded ribonucleic acid (dsRNA) for the manufacture of a medicament for treating a tumor necrosis factor-alpha (TNF- α) -related inflammatory condition in an animal subject, wherein the polynucleotide delivery-enhancing polypeptide is amphipathic and comprises nucleic acid binding properties, wherein the medicament is capable of reducing TNF- α RNA levels, thereby preventing or reducing the occurrence or severity of the TNF- α -related inflammatory condition. In one embodiment, the polynucleotide delivery-enhancing polypeptide 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: 1MW20,000-50,000, poly (Orn, Trp) 4: 1MW20,000-50,000, melittin, histone H1, histone H3 and histone H4, SEQ ID NOS 27-31, 35-42, 45, 47, 50-59, 62, 63, 67, 68, 73, 74, 76, 78-87, 89-92, 94-108, 164-178 and 180-186. In a related embodiment, the N-terminus of the polynucleotide delivery-enhancing polypeptide is acetylated. In another related embodiment, the N-terminus of the polynucleotide delivery-enhancing polypeptide 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 that is complementary to a portion of a tumor necrosis factor-alpha (TNF-alpha) gene. In a related embodiment, the dsRNA is an siRNA consisting of about 10 to about 40 base pair sequence selected from the group consisting of SEQ ID NOS 109-163 and 187. In another embodiment, the polynucleotide delivery-enhancing polypeptide is admixed, complexed or conjugated to the dsRNA. In another embodiment, the polynucleotide delivery-enhancing polypeptide binds to the dsRNA. In another embodiment, the animal subject is a mammal.

Brief description of the drawings

FIG. 1 shows peptide-mediated uptake of siRNAs complexed or conjugated to a polynucleotide delivery-enhancing polypeptide of the present invention (SEQ ID NO: 35) and the effect on cellular activity. Cellular uptake and cellular activity are expressed as a percentage.

FIG. 2 further shows peptide-mediated uptake of siRNAs complexed or conjugated to a polynucleotide delivery-enhancing polypeptide of the present invention (SEQ ID NO: 35). Cellular uptake is expressed as mean fluorescence density (MFI).

Figure 3 shows peptide-mediated uptake of siRNAs and several different polynucleotide delivery-enhancing polypeptides in human monocytes.

FIG. 4 shows the effect on human monocyte activity following exposure to siRNAs complexed with several different polynucleotide delivery-enhancing polypeptides.

Figure 5 shows that RA progression is delayed in siRNA/peptide injected mice compared to RA progression in Ramicade treated subjects. RA progression was measured with paw score index.

Fig. 6 provides the results of studies on the efficacy and activity of PN73 rationally designed derivative polynucleotide delivery-enhancing polypeptides taken up by mouse tail fibroblasts.

Figure 7 shows that peptide-mediated uptake of siRNAs complexed to the polynucleotide delivery-enhancing polypeptides of the invention does not elicit an interferon response compared to liposome-mediated delivery of siRNAs. (A) The method comprises the following steps Sirna complexed with liposome (b): siRNA complexed with PN73 (1: 5).

Figure 8 shows that siRNAs conjugated to a polynucleotide delivery-enhancing polypeptide have greater knockdown activity in vitro than siRNAs complexed to a polynucleotide delivery-enhancing polypeptide.

Fig. 9 shows a comparison of cellular uptake between cholesterol-conjugated siRNAs and unconjugated siRNAs to polynucleotide delivery-enhancing polypeptides.

Figure 10 shows that cholesterol-conjugated siRNAs conjugated to a polynucleotide delivery-enhancing polypeptide are able to rescue serum inhibition of cellular uptake.

Description of exemplary embodiments of the invention

The present invention fulfills these needs and achieves other objects and advantages by providing novel compositions and methods that utilize small interfering ribonucleic acid (siNA), or precursors thereof, in combination with a polynucleotide delivery-enhancing polypeptide. The polynucleotide delivery-enhancing polypeptide is a natural or artificial polypeptide selected for its ability to enhance intracellular delivery or uptake of polynucleotides comprising siNAs and their precursors.

Within the novel compositions of the invention, the siNA can be mixed or complexed or conjugated with a polynucleotide delivery-enhancing polypeptide to form a composition that enhances siNA delivery within a cell, as compared to the delivery resulting from contacting a target cell with a naked siNA (i.e., a siNA without a polynucleotide delivery-enhancing polypeptide of the invention).

In some embodiments of the invention, the polynucleotide delivery-enhancing polypeptide is a histone or polypeptide or peptide fragment, derivative, analog or conjugate thereof. In these embodiments, the siNA is mixed, complexed or conjugated to one or more polypeptides of full-length histone proteins or at least part of the partial sequence of the corresponding histone proteins, such as one or more of the following histone proteins: histone H1, histone H2A, histone H2B, histone H3 or histone H4, or one or more polypeptide fragments or derivatives thereof comprising at least part of the sequence of a histone, typically at least 5-10 or 10-20 contiguous residues of the native histone. In more specific embodiments, the siRNA/histone mixture, complex, or conjugate is substantially free of amphiphilic compounds. In other specific embodiments, the siNA mixed with, complexed or conjugated to a histone or polypeptide comprises double stranded RNA, e.g., double stranded RNA having 30 or fewer nucleotides, and is small interfering RNA (sirna). In exemplary embodiments, the histone polynucleotide delivery-enhancing polypeptide comprises a fragment of histone H2B, such as the polynucleotide delivery-enhancing polypeptide designated PN73 described below. In another embodiment, the polynucleotide delivery-enhancing polypeptide may be pegylated to improve stability and/or efficacy, particularly in the context of in vivo administration.

In further embodiments of the invention, the polynucleotide delivery-enhancing polypeptide is selected or rationally designed to comprise an amino acid sequence that is amphipathic. For example, a useful polynucleotide delivery-enhancing polypeptide can be selected that comprises a plurality of non-polar or hydrophobic amino acid residues that form a hydrophobic sequence domain or motif linked to a plurality of charged amino acid residues that form a charged sequence domain or motif to form an amphipathic peptide.

In other embodiments, the polynucleotide delivery-enhancing polypeptide selected comprises a protein transduction domain or motif, and a fusion-forming peptide domain or motif. A protein transduction domain is a peptide sequence capable of insertion into and preferably across a cell membrane. The fusion-forming peptide is a peptide which destabilizes, for example, a cytoplasmic membrane or a lipid membrane coating an endosomal membrane, and the action of which can be enhanced by low pH. Exemplary fusion-generating domains or motifs can be found in a wide variety of viral fusion proteins and other proteins, such as fibroblast growth factor 4(FGF 4).

In order to rationally design the polynucleotide delivery-enhancing polypeptides of the present invention, the transduction domain of the protein is used as a motif that facilitates entry of the nucleic acid into the cell through the cell membrane. In some embodiments, the delivered nucleic acid is encapsulated within an endosome. The low pH inside the endosome leads to fusion to form a peptide motif that destabilizes the endosome membrane. Destabilization and cleavage of the endosomal membrane allows release of the siNA into the cytosol, where it can bind to the RISC complex and target its target mRNA.

Examples of protein transduction domains of polynucleotide delivery-enhancing polypeptides that are selectively incorporated in the present invention include:

TAT Protein Transition Domain (PTD) (SEQ ID NO: 1) KRRQRRR;

2. cell-penetrating peptide PTD (SEQ ID NO: 2) RQIKIWFQNRRMKWKK;

3.VP22PTD(SEQ ID NO：3)DAATATRGRSAASRPTERPRAPARSASRPRRPVD；

kaposi FGF signal sequence (SEQ ID NO: 4) AAVALLPAVLLALLAP, and SEQ ID NO: 5) AAVLLP LLPVLLAAP, respectively;

5. human β 3 integrin signal sequence (SEQ ID NO: 6) VTVLALGALAGVGVG;

gp41 fusion sequence (SEQ ID NO: 7) GALFLGWLGAAGSTMGA;

7. crocodile of Mikana (Caiman crocodilus) Ig (v) light chain (SEQ ID NO: 8) MGLGLHLLVLAAALQGA;

hCT-derived peptide (SEQ ID NO: 9) LGTYTQDFNKFHTFPQTAIGVGAP;

9.Transportan(SEQ ID NO：10)GWTLNSAGYLLKINLKALAALAKKIL；

10. oligomer (SEQ ID NO: 11) TPPKKKRKVEDPKKKK;

11. arginine peptide (SEQ ID NO: 12) RRRRRRR; and

12. ampholytic pattern peptide (SEQ ID NO: 13) KLALKLALKALKAALKLA.

Examples of viral fusion peptide fusion-generating domains of polynucleotide delivery-enhancing polypeptides selectively incorporated in the present invention include:

1. influenza virus HA2(SEQ ID NO: 14) GLFGAIAGFIENGWEG;

2.Sendai F1(SEQ ID NO：15)FFGAVIGTIALGVATA；

3. respiratory syncytial virus F1(SEQ ID NO: 16) FLGFLLGVGSAIASGV;

HIV gp41(SEQ ID NO: 17) GVFVLGFLGFLATAGS; and

5. eparovirus GP2(SEQ ID NO: 18) GAAIGLAWIPYFGPAA.

In further embodiments of the invention, polynucleotide delivery-enhancing polypeptides are provided that incorporate a DNA-binding domain or motif that, within the methods and compositions of the invention, facilitate the formation of polypeptide-siNA complexes and/or enhance the delivery of siNAs. DNA binding domains exemplified herein include the "zinc finger" domains described as DNA binding regulatory proteins and other proteins identified in Table 1 below (see, e.g., Simpson, et al., J.biol.Chew, 278: 28011-.

^*This table shows conserved zinc finger motifs that bind double stranded DNA, characterized by a 0-x (2, 4) -C-x (12) -H-x (3) -H motif pattern (SEQ E) NO: 188) it can be used to select and design additional polynucleotide delivery-enhancing polypeptides according to the invention itself.

^**The sequences of Sp1, Sp2, Sp3, Sp4, DrosBtd, DrosSp, cet22c8.5 and y4pb1a.4 shown in table 1 are designated in the present invention as SEQ ID NO: s 19, 20, 21, 22, 23, 24, 25 and 26.

Other DNA binding domains useful in constructing a polynucleotide delivery-enhancing polypeptide of the invention include, for example, portions of the HTV Tat protein sequence (see examples below).

In the exemplary embodiments of the invention described below, polynucleotide delivery-enhancing polypeptides are rationally designed and constructed to effectively mediate enhanced delivery of siNAs into target cells by incorporating any of the aforementioned structural elements, domains or motifs into a single-chain polypeptide. For example, the TAT polypeptide protein transduction domain is fused to the N-terminal 20 amino acids of the influenza hemagglutinin protein designated HA2, resulting in the exemplary polynucleotide delivery-enhancing polypeptides of the present invention. Numerous other polynucleotide delivery-enhancing polypeptide constructs are provided in the present disclosure, demonstrating the broad application of the concepts of the present invention to efficient polynucleotide delivery-enhancing polypeptides that generate and use a diverse assembly of enhanced siNA delivery.

Further exemplary polynucleotide delivery-enhancing polypeptides of the invention may be selected from the following peptides: WETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27); GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO: 29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), poly Lys-Trp, 4: 1, MW20,000 and 50,000; and poly Orn-Trp, 4: 1, MW20,000 and 50,000. Additional polynucleotide delivery-enhancing polypeptides for use in the compositions and methods of the invention comprise all or part of a melittin protein sequence.

Still other exemplary polynucleotide delivery-enhancing polypeptides are identified in the examples below. Within the methods and compositions of the invention, any one or combination of these peptides can be selected or combined to produce effective polynucleotide delivery-enhancing polypeptide agents, to induce or facilitate intracellular delivery of siNAs.

In more specific aspects of the invention, a mixture, complex or conjugate comprising an siRNA and a polynucleotide delivery-enhancing polypeptide may optionally be combined with, for example, LIPOFECTIN liposomesThe cationic lipid junctions of (e.g., mixed or complexed). Herein, it is unexpectedly disclosed that a polynucleotide delivery-enhancing polypeptide complexed or conjugated to siRNA alone itself can accomplish delivery of the siNA sufficient to mediate gene silencing of RNAi. However, it is further unexpected that complexes or conjugates of siRNA/polynucleotide delivery-enhancing polypeptides exhibit greater activity in mediating siNA delivery and gene silencing when mixed or complexed with cationic lipids, such as liposomes. To prepare these complexes consisting of the polynucleotide delivery-enhancing polypeptide, the siRNA and the cationic lipid, the siRNA and the peptide can be first mixed together in a suitable medium, such as a cell culture medium, after which the cationic lipid is added to the mixture to form the siRNA/delivery peptide/cationic lipid complex. Optionally, the siRNA and cationic lipid can be first mixed together in a suitable medium, such as a cell culture medium, after which the siRNA is added to the mixture to form an siRNA/delivery peptide/cationic lipid complex.

Examples of cationic lipids useful in these aspects of the invention include N- [ 1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride, 1, 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane, 1, 2-bistetradecyloxypropropan3-bis-methyl-hydroxyethylammonium bromide, bis-octadecyl-dimethylammonium bromide, 2, 3-dioleoyloxy-N- [ 2- (spermine-carboxamido) ethyl]ammonium-N, N-dimethyl-1-propyltrifluoroacetate, 1, 3-dioleoyloxy-2- (6-carboxyspermine) -propionamide, 5-carboxysperminamine aminoacetic acid dioctadecylamide, tetramethyltetrapalmitospermine, tetramethyltetraoleospermine, tetramethyltetralauryl spermine, tetramethyltetramyristyl spermine and tetramethyldioleospermine, DOTMA (N- [ 1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane), DMRIE (1, 2-bistetradecyloxypropyl-3-dimethylhydroxyethylammonium bromide), DDAB (dioctadecyldimethylammonium bromide), Polyvalent cation liposome, lipospermine, DOSPA (2, 3-dioleoyloxy-N- [2 (spermine carboxamide) ethyl)]-ammonium N, N-dimethyl-1-propyltrifluoroacetate), DOSPER (1, 3-dioleoyloxy-2- (6-carboxyspermine) -propionamide, di-and tetra-alkyl-tetra-methyl spermine), TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleoyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMS (tetramethyltetramyristyl spermine), TMDOS (tetramethyldioleoyl spermine) DOGS (dioctadecylamidoglycyl spermine (TRANSFECTAM)). Other useful cationic lipids are described, for example, in U.S. patent No. 6,733,777, U.S. patent No. 6,376,248, U.S. patent No. 5,736,392, U.S. patent No. 5,686,958, U.S. patent No. 5,334,761, and U.S. patent No. 5,459,127.

Cationic lipids are optionally combined with non-cationic lipids, in particular neutral lipids, for example lipids such as DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol. Consists of a 3: 1(w/w) mixture of DOSPA and DOPE and DOTMA and DOPE (LIPOFECTIN)Invitrogen) is commonly used in the compositions of the invention for transfection. Superior foodThe transfection compositions of choice are those which induce sufficient transfection of higher eukaryotic cell lines.

In exemplary embodiments, the present invention features compositions comprising small nucleic acid molecules, such as short interfering nucleic acids (siNA), short interfering rnas (sirna), double stranded rnas (dsrna), micrornas (mrna), or short hairpin rnas (shrna), mixed, complexed or conjugated to a polynucleotide delivery-enhancing polypeptide.

The term "short interfering nucleic acid", "siNA", "short interfering RNA", "siRNA", "short interfering nucleic acid molecule" or "chemically modified short interfering nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of inhibiting or down-regulating gene expression or viral replication, e.g., by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. In exemplary embodiments, the siNA is a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleic acid sequence, or portion thereof, that is complementary to a nucleotide sequence of a target nucleic acid molecule that downregulates expression, and the sense region comprises a nucleic acid sequence that corresponds to (i.e., is substantially identical in sequence to) the target nucleic acid sequence, or portion thereof.

"siNA" refers to a small interfering nucleic acid, such as siRNA, that is a double-stranded nucleic acid of short length (or optionally, a longer precursor thereof) that is not unacceptably toxic within the target cell. The length of siNAs useful in the present invention is in some embodiments optimized at a length of about 21-23 bp. However, the length of useful siNAs including siRNAs is not particularly limited. For example, siNAs can be initially presented to a cell in a precursor form that is substantially different from a terminal or processed form of the siNA that is present and exerts gene silencing activity upon or after delivery to a target cell. Precursor forms of siNAs, for example, include precursor sequence elements that are processed, degraded, altered, or cleaved upon or after delivery to produce intracellular active siNAs that mediate gene silencing. Thus, in some embodiments, siNAs useful within the invention have, for example, a precursor length of about 100-200 base pairs, 50-100 base pairs, or less than about 50 base pairs, which results in an active, processed siNA within the target cell. In other embodiments, useful siNA or siNA precursors are about 10 to 49bp, 15 to 35bp, or about 21 to 30bp in length.

As described above, in certain embodiments of the invention, polynucleotide delivery-enhancing polypeptides are used to facilitate delivery of nucleic acid molecules, including large siNAs nucleic acid precursors, that are larger than conventional siNAs. For example, the methods and compositions of the invention can be used to enhance delivery of larger nucleic acids representing "precursors" to a siNAs of interest, wherein the precursor amino acids can be cleaved or processed before, during, or after delivery to the target cell to form active siNAs that modulate gene expression within the target cell. For example, a siNA precursor polynucleotide can be selected as a circular, single-stranded polynucleotide comprising a self-complementary sense region and antisense region, wherein the antisense region comprises a nucleic acid sequence complementary to a target nucleic acid molecule or portion thereof, the nucleotide sequence of the sense region corresponds to the target nucleic acid sequence or portion thereof, and a stem portion, wherein the circular polynucleotide can be processed in vivo or in vitro to produce an active siNA molecule capable of mediating RNAi.

In mammalian cells, dsRNAs longer than 30 base pairs activate the dsRNA dependent kinases PKR and 2', 5-oligoadenylate synthetase, which are normally induced by interferon. Activated PKR suppresses general translation by phosphorylating translation transcription factors initiating the eukaryotic promoter 2 α (eIF2 α), while 2', 5-oligoadenylate synthetase leads to non-specific niRNA degradation by activating RNase L. The siNAs described in the present invention avoid the activation of interferon response due to their small size (especially in the non-precursor form), usually less than 30 base pairs, most usually in the range of about 17-19, 19-21 or 21-23 base pairs.

Compared to the nonspecific effect of long dsrnas, sirnas can mediate selective gene silencing in mammalian systems. Hairpin RNAs with a short loop and 19 to 27 base pairs in the stem also selectively silence the expression of genes complementary to sequences on their double-stranded stem. Mammalian cells can convert short hairpin RNAs into sirnas to mediate selective gene silencing.

RISC mediates cleavage of single-stranded RNA containing sequences complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex. Studies have shown that 21 nucleotides of siRNA duplex activity is highest when it contains a2 nucleotide overhang at the 3' end. Furthermore, complete substitution of one or both siRNA strands with 2 ' -deoxy (2 ' -H) or 2 ' -O-methyl nucleotides abolished RNAi activity, whereas substitution of the nucleotide overhanging the end of siRNA3 ' with deoxy nucleotide (2 ' -H) was reported to be tolerated.

Studies have shown that substitution of deoxynucleotides for the 3 'overhang fragment of the 21-mer siRNA duplex with 2 3' nucleic acid overhangs has no detrimental effect on RNAi activity. It has been reported that substitution of up to 4 nucleosides per end of the siRNA with deoxynucleotides is well tolerated, whereas complete substitution with deoxynucleotides results in loss of RNAi activity.

Alternatively, the siNAs may be delivered as single or multiple transcripts expressed from a polynucleotide vector encoding the single or multiple siNAs and directing their expression in the target cell. In these embodiments, the length of the double stranded portion of the final transcription product of siRNAs expressed in the target cell may be, for example, 15 to 49bp, 15 to 35bp, or about 21 to 30 bp. In exemplary embodiments, the double-stranded portion of the siNAs (where both strands are paired) is not limited to a perfectly paired nucleoside fragment, and may include unpaired portions due to mismatches (corresponding nucleotides are not complementary), bulges (lack of corresponding complementary nucleotides on one strand), overhangs, and the like. The extent to which the unpaired parts can be included is that they do not interfere with siNA formation. In more detailed embodiments, a "bulge" can comprise 1 to 2 unpaired nucleotides, and a two-strand paired siNAs double-stranded region can comprise a bulge from about 1 to 7 or about 1 to 5. In addition, the number of "mismatch" moieties contained in the siNAs double-stranded region that can occur is from about 1 to 7 or about 1 to 5. The most susceptible mismatched nucleotides are guanine and uracil. These mismatches can be attributed, for example, to C to T, G to A mutations in the corresponding DNA encoding the sense RNA, but other reasons are also contemplated. Furthermore, in the present invention, the siNAs double-stranded region of the two-strand pair may contain both bulge and mismatch portions within a specified approximate number range.

The terminal structure of siNAs in the present invention can be blunt or cohesive (protruding) as long as the siNA retains its activity to silence target gene expression. The sticky (protruding) end structure is not limited to the 3' protrusion reported by others. Rather, a 5' overhang structure can be included so long as it can induce a gene silencing effect, e.g., via RNAi. Furthermore, the number of protruding nucleotides is not limited to the reported 2 or 3 nucleotide limit, but can be any number, as long as the protrusion does not disrupt the gene silencing activity of the siNA. For example, an overhang may comprise from 1 to 8 nucleotides, more typically from 2 to 4 nucleotides. The total length of the siNAs with sticky end structures is expressed as the sum of the length of the paired double stranded parts and the length of the pair of single strands comprising the overhang at both ends. For example, in the case of a19 bp double-stranded RNA with a 4-nucleotide overhang at both ends, the total length is represented as 23 bp. Furthermore, because the overhang sequence may have low specificity for the target gene, it is not necessarily complementary (antisense) or identical (sense) to the target gene sequence. Furthermore, the siNA may comprise low molecular weight structures (e.g., natural RNA molecules such as tRNA, rRNA, or viral RNA, or artificial RNA molecules), e.g., overhangs at the ends, as long as it maintains its gene silencing effect on the target gene.

Furthermore, the end structure of siNAs may have a stem-loop structure, wherein one end of the double-stranded nucleic acid is linked by a linker nucleic acid, e.g., a linker RNA. The length of the double-stranded region (stem-loop portion) may be, for example, 15 to 49bp, often 15 to 35bp, more usually about 21 to 30 bp. Alternatively, the double-stranded region of the final transcript of siNAs expressed in the target cell may be, for example, about 15 to 49bp, 15 to 35bp, or about 21 to 30bp in length. When a linker fragment is used, there is no particular limitation on the length of the linker, as long as it does not interfere with the pairing of the stems. For example, the linker moiety may have a clover tRNA structure in order to stabilize the pairing of stems and inhibit recombination between DNAs encoding the moiety. Even if the length of the linker prevents pairing of the stems, it is possible to allow pairing of the stems, for example, to construct a linker part containing an intron that is cut off during processing of the precursor RNA into mature RNA, thereby allowing pairing of the stems. In stem-loop siRNAs, RNA without a loop structure can have low molecular weight RNA at either end (head or tail). As described above, these low molecular weight RNAs may include natural RNA molecules such as tRNA, rRNA or viral RNA, or artificial RNA molecules.

A siNA can also comprise a single-stranded polynucleotide comprising a nucleotide sequence that is complementary to a nucleotide sequence of a target nucleic acid molecule or portion thereof (e.g., such a siNA molecule need not have a nucleotide sequence corresponding to a target nucleic acid sequence or portion thereof present in the siNA molecule), wherein the single-stranded polynucleotide can further comprise a terminal phosphate group, such as a 5' -phosphate group (see, e.g., Martinez, et al, cell.110: 563-.

As used herein, the term siNA molecule is not limited to containing only naturally occurring RNA or DNA, but also includes chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack nucleotides containing a2 '-hydroxyl group (2' -OH). In certain embodiments, short interfering nucleic acids do not require the presence of nucleotides containing a2 '-hydroxyl group to mediate RNAi, and as such, short interfering nucleic acid molecules of the invention may optionally not include any ribonucleotides (e.g., nucleotides containing a 2' -hydroxyl group). However, these siNA molecules that do not require the presence of ribonucleotides in the siNA molecule to support RNAi may have an attachment linker or linkers or other attached or associated groups, moieties or strands comprising one or more nucleotides with a 2' -hydroxyl group. Optionally, the siNA molecule may comprise ribonucleotides at about 5%, 10%, 20%, 30%, 40% or 50% of the nucleotide positions.

The term siNA as used herein is intended to be equivalent to other terms used to describe nucleic acid molecules capable of mediating sequence-specific RNAi, such as short interfering rna (siRNA), double-stranded rna (dsrna), microrna (mrna), short hairpin rna (shrna), short interfering oligonucleotides, short interfering nucleic acids, short interference-modified oligonucleotides, chemically-modified sirnas, post-transcriptional gene-silencing rna (ptgsrna), and others.

In other embodiments, siNA molecules used in the invention may comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleoside or non-nucleoside linker molecules, or alternatively non-covalently linked by ionic interactions, hydrogen bonds, fanning force interactions, hydrophobic interactions, and/or stacking interactions.

In other embodiments, siNA molecules used in the invention may comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleoside or non-nucleoside linking molecules, or alternatively, are non-covalently linked by ionic interactions, hydrogen bonds, van der waals interactions, hydrophobic interactions, and/or stacking (stacking) interactions.

"antisense RNA" refers to an RNA strand having a sequence complementary to the mRNA of a target gene, and is thought to induce RNAi by binding to the mRNA of the target gene. A "sense RNA" has a sequence complementary to an antisense RNA that anneals to its complementary antisense RNA and hybridizes to form an siRNA. These antisense and sense RNAs are routinely synthesized by RNA synthesizers.

The term "RNAi construct" as used in the present invention is a generic term used in the present specification and includes small interfering RNAs (siRNAs), hairpin RNAs and other RNA types that can be cleaved in vivo to form siRNAs. The RNAi constructs of the invention also include expression vectors (also referred to as RNAi expression vectors) capable of producing transcripts of dsRNAs or hairpin RNAs within a cell and/or siRNAs in vivo. Optionally, the siRNA comprises single or double stranded siRNA.

siHybrid molecules are double-stranded nucleic acids with similar functions as siRNA. sihybrids consist of both RNA and DNA strands, rather than double-stranded RNA molecules. Preferably, the RNA strand is the antisense strand, the same strand as that bound to the target mRNA. Sihybrids produced by hybridization of a DNA strand and an RNA strand have a hybridization complementary portion and preferably at least one 3' overhanging end.

The siNAs used in the present invention may be assembled from two separate oligonucleotides, one of which is the sense strand and the other of which is the antisense strand, wherein the antisense and sense strands are themselves complementary (i.e. each strand contains a nucleotide sequence complementary to the nucleotide sequence of the other strand; such that the antisense and sense strands therein form a double-stranded or double-stranded structure, e.g. wherein the double-stranded region is about 19 base pairs). The antisense strand contains a nucleotide sequence that is complementary to a nucleotide sequence of the target nucleic acid molecule or a portion thereof, and the sense strand may contain a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the siNA may be assembled from a single oligonucleotide in which sense and antisense regions of self-complementarity of the siNA are linked by a nucleic acid-based or non-nucleic acid-based linker.

In another embodiment, according to the methods and compositions of the present invention, the siNAs for intracellular delivery may be a double-stranded, asymmetric double-stranded, hairpin-containing or asymmetric hairpin secondary structure polynucleotide having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to an isolated target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.

Non-limiting examples of chemical modifications that occur in siNA include, but are not limited to, phosphorothioate internucleotide linkages (phosphorothioate internucleotide linkages), 2 '-deoxynucleotides, 2' -O-methyl nucleotides, 2 '-deoxy-2' -fluoro nucleotides, "universal base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and incorporation of terminal glyceryl and/or inverted deoxyabasic residues. It has been shown that these chemical modifications, when used in various siNA constructs, can protect intracellular RNAi activity while significantly increasing the plasma stability of these compounds.

In a non-limiting example, the introduction of chemically modified nucleotides into nucleic acid molecules provides a powerful tool for overcoming potential limitations of in vivo stability and biological activity inherent in exogenously delivered native RNA molecules. For example, because chemically modified nucleic acid molecules tend to have longer half-lives in serum, the use of chemically modified nucleic acid molecules enables a given therapeutic effect to be achieved with lower doses of a particular nucleic acid molecule. Moreover, certain chemical modifications can increase the biological activity of a nucleic acid molecule by targeting specific cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Thus, even if the activity of the chemically modified nucleic acid molecule is reduced compared to the native nucleic acid molecule, e.g., compared to a full-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule is higher than the activity of the native molecule due to the improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically modified siNA also minimizes the possibility of activating human interferon.

In siNA molecules described herein, the antisense region of siNA molecules of the invention includes phosphorothioate internucleotide linkages at the 3' terminus of the antisense region.

In any of the embodiments of siNA molecules described herein, the antisense region comprises from about 1 to about 5 phosphorothioate internucleotide linkages at the 5' terminus of the antisense region described above. In any of the embodiments of siNA molecules described herein, the 3' terminal nucleotide overhang of a siNA molecule of the invention can comprise a ribonucleic acid or a deoxyribonucleic acid chemically modified on the nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3' terminal nucleotide protuberance can comprise one or more universal base nucleotides. In any of the embodiments of siNA molecules described herein, the 3' terminal nucleotide protuberance can comprise one or more acyclic nucleotides.

For example, in a non-limiting example, the invention features chemically modified short interfering nucleic acids (sinas) having 1, 2, 3, 4, 5,6, 7, 8, or more phosphorothioate internucleotide linkages in one siNA strand. In another embodiment, the invention features chemically modified short interfering nucleic acids (sinas) having 1, 2, 3, 4, 5,6, 7, 8, or more phosphorothioate internucleotide linkages in each of the two siNA strands. Phosphorothioate internucleotide linkages may be present in one or both oligonucleotide strands in the siNA double strand, for example in the sense strand, the antisense strand or both strands. The siNA molecules of the invention may comprise one or more phosphorothioate internucleotide linkages at the 3 '-end, the 5' -end, or both the 3 '-end and the 5' -end of the sense strand, antisense strand, or both strands. For example, a siNA exemplified in the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5 or more consecutive phosphorothioate internucleotide linkages at the 5' -terminus of the sense, antisense, or both strands. In another non-limiting example, a siNA molecule exemplified herein can comprise one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more consecutive) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more consecutive) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.

A siNA molecule can consist of a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length, about 18 to about 23 base pairs (e.g., about 18, 19, 20, 21, 22, or 23), wherein the circular oligonucleotide forms a dumbbell structure with about 19 base pairs and 2 loops.

A cyclic siNA molecule comprises two loop motifs, wherein one or both loop portions of the siNA molecule are biodegradable. For example, the circular siNA molecules of the invention are designed such that in vivo degradation of the loop portion of the siNA molecule can produce a double-stranded siNA molecule with a3 '-terminal protrusion, e.g., a 3' -terminal nucleotide protrusion containing 2 nucleotides.

The modified nucleotides present in the siNA molecule, preferably in the antisense strand of the siNA molecule, may also be selected to comprise modified nucleotides having properties or characteristics similar to those of naturally occurring ribonucleotides, in the sense strand and/or in both the antisense and sense strands. For example, siNA molecules that include modified nucleotides that have a Northern conformation (e.g., a Northern pseudo-turn cycle, see, e.g., Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984) are described. As such, the siNA molecules present in the invention, preferably the antisense strand of the siNA molecules in the invention, may also be selected to be resistant to nuclease degradation by chemically modified nucleotides in the sense strand and/or in both the antisense and sense strands, while maintaining the ability to mediate RNAi. Non-limiting examples of nucleotides having the northern configuration include Locked Nucleic Acid (LNA) nucleotides (e.g., 2 '-O, 4' -C-methylene- (D-ribofuranosyl) nucleotides); 2' -Methoxyethoxy (MOE) nucleotide; 2 ' -methyl-thio-ethyl, 2 ' -deoxy-2 ' -fluoro nucleotide; 2 '-deoxy-2' -chloronucleotide, 2 '-azido nucleotide, and 2' -O-methyl nucleotide.

The sense strand of a double-stranded siNA molecule can contain a terminal cap moiety, e.g., an inverted deoxybase moiety, at the 3 '-end, the 5' -end, or both the 3 '-end and the 5' -end of the sense strand.

Non-limiting examples of conjugates include the conjugates and ligands described in U.S. application No. 10/427,160, filed on 30/4/2003 in varesee, et al, which is incorporated herein by reference in its entirety, including the drawings. In another embodiment, the conjugate is covalently attached to the chemically modified siNA molecule via a biodegradable linker. In one embodiment, the coupling molecule is bound to the 3' -terminus of the sense strand, antisense strand, or both strands of the chemically modified siNA molecule. In another embodiment, the coupling molecule is bound to the 5' -terminus of the sense strand, the antisense strand, or both strands of the chemically modified siNA molecule. In another embodiment, the coupling molecule is bound to the 3 '-end and the 5' -end of the sense strand, the antisense strand, or both strands of the chemically modified siNA molecule or any combination thereof. In one embodiment, the coupling molecules of the invention comprise molecules that facilitate the delivery of chemically modified siNA molecules into biological systems, such as cells. In another embodiment, the conjugate that binds to the chemically modified siNA molecule is polyethylene glycol, human serum albumin, or a ligand of a cellular receptor that mediates uptake by cells. Examples of specific coupling molecules that can bind to chemically modified siNA molecules to which the present invention relates are described in varesese et al, U.S. patent application publication No. 20030130186 published on 10.7.2003 and U.S. patent application publication No. 20040110296 published on 10.7.2004. The type of conjugate used in the invention and the extent of conjugation of the siNA molecule are evaluated with improved pharmacokinetic profiles, bioavailability, and/or stability of the siNA construct, while maintaining the ability of the siNA to mediate RNAi activity. Thus, one skilled in the art can screen siNA constructs modified with various conjugates to determine whether a siNA conjugate complex possesses improved properties while retaining the ability to mediate RNAi, for example in animal models generally known in the art.

The siNA may further comprise a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker connecting the siNA sense region to the siNA antisense region. In one embodiment, the nucleotide linker may be > 2 nucleotides in length, for example about 3, 4, 5,6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the nucleotide linker may be a nucleic acid aptamer (aptamer). As used herein, an "aptamer" or "nucleic acid aptamer" refers to a nucleic acid molecule that specifically binds to a target molecule, wherein the sequence of the nucleic acid molecule comprises a sequence that is recognized by the target molecule in its natural environment. Alternatively, the aptamer may be a nucleic acid molecule that binds to a target molecule, wherein the target molecule does not naturally bind to the nucleic acid. The target molecule may be any molecule of interest. For example, aptamers can be used to bind to the ligand binding domain of a protein, thereby preventing the interaction of naturally occurring ligands with the protein. This is a non-limiting example and those skilled in the art will recognize that other embodiments are readily produced using techniques well known in the art (see, e.g., 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; Yasser, J.Biotechnol. 74: 21, 2000; Hermann and Patel, Science 287: 820, 2000; and Jayasena, Clinical Chemistry 45: 1628, 1999).

The non-nucleotide linker may be composed of abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, hydrocarbons or other polymeric compounds (e.g., polyethylene glycols such as those having 2 to 100 ethylene glycol units). Specific examples include those described by Seelaand Kaiser, Nucleic Acids Res.18: 6353, 1990, and Nucleic Acids Res.75: 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, Nucleotides & Nucleotides 10: 281, 1991; jschke, et al, Tetrahedron lett.34: 301, 1993; ono, et al, Biochemistry 30: 9914, 1991; arnold, et al, international publication No. WO 89/02439; usman, et al, international publication No. WO 95/06731; dudycz, et al, international publication nos. wo 95/11910and Ferentz and Verdine, j.am.chem.soc.113: 4000, 1991. "non-nucleotide" further refers to any group or compound that can be incorporated into a nucleic acid strand at the position of one or more nucleic acid units, including sugar and/or phosphate substitutes, and can allow the remaining bases to exert their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base such as adenine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.

The synthesis of the siNA molecules that can be chemically modified in the present invention comprises: (a) synthesizing two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, the two complementary strands of the siNA molecule are synthesized by solid phase oligonucleotide synthesis (solid phase oligonucleotide synthesis). In another embodiment, the two complementary strands of the siNA molecule are synthesized by solid phase tandem oligonucleotide synthesis (solid phase tandem oligonucleotide synthesis).

Oligonucleotides (e.g., certain modified oligonucleotides or oligonucleotide moieties lacking ribonucleotides) are synthesized using protocols known in the art, for example, as described in carothers, et al, methods in Enzymology 211: 3-19, 1992; thompson, et al, international PCT application publication No. WO 99/54459; wincott, et al, Nucleic Acids Res.25: 2677-2684, 1995; wincott, et al, Methods moi.bio.74: 59, 1997; brennan, et al, Biotechnol bioeng.61: 33-45, 1998; and Brennan, as described in U.S. patent No. 6,001,311. The synthesis of RNA, including certain siNA molecules of the invention, follows, for example, the methods described in Usman, et al, j.am.chem.soc.109: 7845, 1987; scaring, et al, Nucleic Acids res.18: 5433, 1990; and Wincott, et al, Nucleic Acids res.23: 2677-2684, 1995; wincott, et al, Methods moi.bio.74: 59, 1997.

For example, in Akhtar, et al, Trends Cell bio.2: 139, 1992; delivery strategies for Antisense Oligonucleotide Therapeutics, ed. akhtar, 1995; maurer, et al, moi.membr.biol.16: 129-140, 1999; hofling and Huang, handb. exp. pharmacol.137: 165-192, 1999; and Lee, et al, ACS symp.ser.752: 184-192, 2000 describe complementary or complementary methods for delivering nucleic acid molecules for use in the present invention. Sullivan, et al, International PCT publication No. WO 94/02595 further describes general methods for enzyme nucleic acid molecule delivery. These protocols can be used to complement or complement the delivery of almost any nucleic acid molecule involved in the present invention.

Nucleic acid molecules and polynucleotide delivery-enhancing polypeptides can be administered to cells by a variety of methods known to those of skill in the art, including, but not limited to, administration within a formulation that includes only siNA and the polynucleotide delivery-enhancing polypeptide, or that further includes one or more additional ingredients, such as pharmaceutically acceptable carriers, diluents, excipients, adjuvants, emulsifiers, buffers, stabilizers, preservatives, and the like. In certain embodiments, siNA and/or polynucleotide transport-enhancing polypeptides can be encapsulated within liposomes, administered by iontophoresis, or combined with, for example, a hydrogel, a cyclodextrin, a biodegradable nanocapsule, a bioadhesive microsphere, or a protein carrier (see, e.g., O' Hare and Normand, international PCT publication No. WO 00/53722). Alternatively, the nucleic acid/peptide/vector composition may be administered locally by direct injection or using an infusion pump. Using standard needle and syringe methodologies or as described, for example, in Conry, et, clin. 2330-.

The composition of the present invention can be effectively used as a pharmaceutical agent. The pharmaceutical agent prevents, modulates the occurrence or severity of, or treats (alleviates to a monitorable or quantifiable extent one or more symptoms) the disease state or other adverse condition of the patient.

Thus in additional embodiments, the invention provides pharmaceutical compositions and methods characterized by the presence or administration of one or more polynucleic acids, typically one or more siNAs, combined, complexed or conjugated to a polynucleotide delivery-enhancing polypeptide, optionally in combination with a pharmaceutically acceptable carrier formulation such as diluents, stabilizers, buffers, and the like.

The present invention meets other objects and advantages by providing short interfering nucleic acid (siNA) molecules that modulate the expression of genes associated with a particular disease state or other adverse state in a subject. Typically, the siNA targets a gene that is highly expressed as a factor contributing to or acting in association with a disease state or adverse state in the subject. Herein, the siNA effectively down-regulates the gene to a level that prevents, alleviates, or reduces the severity or recurrence of one or more symptoms of the associated disease. Alternatively, expression of a target gene is not necessarily increased as a result or outcome of a disease or other adverse condition for various disease models, but downregulation of the target gene produces a therapeutic effect by decreasing gene expression (i.e., decreasing the level of selected mRNA and/or protein product of the target gene). Alternatively, siNAs in the present invention are optionally targeted to reduce the expression of one gene, resulting in the up-regulation of a "downstream" gene whose expression is inversely regulated by the product or activity of the target gene.

In exemplary embodiments, the compositions and methods of the present invention are useful as therapeutic tools for modulating tumor necrosis factor-alpha (TNF- α) expression for treating or preventing the symptoms of Rheumatoid Arthritis (RA). The invention further provides herein compounds, compositions and methods for modulating the expression and activity of TNF-alpha via RNA interference (RNAi) of small nucleic acid molecules. In more detailed embodiments, the invention provides small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering rna (sirna), double-stranded rna (dsrna), micro-rna (mrna), and short hairpin rna (shrna) molecules, and related methods, that are effective for modulating expression of TNF-a and/or TNF-a genes to prevent or reduce RA symptoms in a mammalian subject. In these and related therapeutic compositions and methods, the use of chemically modified siNAs, for example, by providing increased resistance to nuclease degradation in vivo and/or by improved cellular uptake, generally enables improved properties of the modified siNAs compared to the properties of the native siNA molecule. Useful siNAs with multiple chemical modifications will retain their RNAi activity, as can be readily determined from the disclosure of the present invention. Thus, the siNA molecules of the invention provide useful reagents and methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

The siNAs of the invention may be administered in any form, for example transdermally or by local injection (e.g. locally at the site of psoriatic plaques to treat psoriasis, or injected into the joints of patients suffering from psoriatic arthritis or RA). In a more detailed embodiment, the present invention provides formulations and methods for administering therapeutically effective doses of siNAs against TNF- α mRNA that are effective to down-regulate the TNF- α RNA and thereby reduce or prevent one or more TNF- α -related inflammatory states. The present invention provides relative methods and compositions for targeting the expression of one or more genes associated with a selected disease state in an animal subject, including any of a number of genes whose expression is aberrantly increased as a factor known to be responsible for or contributing to the selected disease state.

The siNA/polynucleotide delivery-enhancing polypeptide mixture of the invention may be administered in combination with other standard treatments for the target disease state, for example in combination with therapeutic agents effective against inflammatory diseases such as RA or psoriasis. Examples of useful and effective agents for combination herein include non-steroidal anti-inflammatory drugs (NSAIDs), methotrexate, gold compounds, D-penicillamine, antimalarials, sulfasalazine, glucocorticoids, and other TNF- α neutralizing agents such as infliximab and entracept.

The negatively charged polynucleotides (e.g., RNA or DNA) of the invention can be administered to a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When a liposome delivery mechanism is desired, standard protocols for forming liposomes can be followed. The compositions of the present invention may also be formulated for use as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions for injection, suspensions and other compositions known in the art.

The invention also includes pharmaceutically acceptable formulations of the compositions described herein. These formulations include salts of the above compounds, such as acid addition salts, e.g., hydrochloride, hydrobromide, acetate and benzenesulfonate salts.

A pharmaceutical composition or formulation refers to a composition or formulation in a form suitable for administration (e.g., systemic administration) to a cell or patient, including, for example, a human. Suitable forms depend in part on the use or route of administration, e.g., oral, transdermal or injection. These forms should not prevent the composition or formulation from reaching the target cell (i.e., the cell to which the negatively charged nucleic acid is to be delivered). For example, a pharmaceutical composition injected into the bloodstream should be soluble. Other factors are known in the art, including considerations such as toxicity.

By "systemic administration" is meant the systemic absorption or accumulation of a drug in the bloodstream in the body, followed by distribution throughout the body. Routes of administration that result in systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, and intramuscular. Each of these routes of administration exposes negatively charged polymers, e.g., nucleic acids, to diseased tissue that is accessible. The rate of drug entry into the circulation has been shown to be a function of molecular weight or volume. The use of liposomes and other drug carriers comprising the compounds of the invention can potentially localize drugs to, for example, certain tissue types such as reticuloendothelial system (RES) tissues. Liposomal formulations that facilitate the binding of drugs to cell surfaces such as lymphocytes and macrophages are also useful. This approach can provide enhanced delivery of drugs to target cells by exploiting the immunological recognition specificity of macrophages and lymphocytes for abnormal cells such as cancer cells.

By "pharmaceutically acceptable formulation" is meant a composition or formulation that allows for the efficient distribution of the nucleic acid molecules of the invention in the physiological location most suitable for their desired activity. Non-limiting examples of reagents suitable for formation using the nucleic acid molecules of the invention include: p-glycoprotein inhibitors (e.g., polyether P85) that enhance drug entry into the central nervous system (Jolliet-Riant and Tillement, fundam. Clin. Pharmacol.13: 16-26, 1999); biodegradable polymers such as lactic-co-glycolic acid microspheres for sustained release delivery of agents following intracerebral transplantation (Emerich, d.f., et al, Cell Transplant 5: 47-58, 1999) (Alkermes, inc. cambridge, Mass); and nanoparticles such as those composed of poly-butyl- α -cyanoacrylate, which are capable of transporting drugs across the blood-brain barrier and altering neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry 25: 941-949, 1999). Other non-limiting examples of nucleic acid molecule delivery strategies for use in the present invention include those described in Boado, et al, j.pharm.sd.57: 1308 charge 1315, 1998; tyler, et al, FEBS lett.427: 280-284, 1999; pardridge, et al, PNAS usa.92: 5592-5596, 1995; boado, adv. drug delivery rev.75: 73-107, 1995; Aldrian-Herrada, et al, Nucleic Acids Res.26: 4910 4916, 1998; and Tyler, et al, PNAS usa.. 96: 7053-7058, 1999.

The invention also includes compositions for storage or administration comprising a pharmaceutically effective dose of the desired compound in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's pharmaceutical Sciences, Mack Publishing co., a.r. gennaro ed., 1985. For example, preservatives, stabilizers, drying agents and flavoring agents may be provided. These include sodium benzoate, sorbic acid and p-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.

A pharmaceutically effective dose is a dose required to prevent, inhibit the appearance of, or treat (alleviate to some extent one, preferably all, symptoms) a disease state. The pharmaceutically effective dose will depend on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physiological characteristics of the particular mammal being considered, the co-administration and other factors recognized by those skilled in the art. In general, the active ingredient is administered at a dose of 0.1mg/kg and 100mg/kg body weight/day, 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, carboxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene oxide cetyl alcohol (heptadecaethylene oxide), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol alcohols, for example polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. These aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoic acid, one or more colour developing agents, one or more flavouring agents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient 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 oil suspension may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavouring agents may be added to provide a palatable oral preparation. Antioxidants such as ascorbic acid may be added to preserve these compositions.

Bulk powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents have already been mentioned above. Other excipients, such as sweeteners, additives and colour developers, may also be present.

The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil or a mineral oil or a mixture of both. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and inositol, anhydrides, for example sorbitan monooleate, and condensation products of the above partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. These emulsifiers may also contain sweeteners and additives.

The pharmaceutical compositions may be in the form of aqueous or oily suspensions for sterile injection. The suspension may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents as mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic miscible diluent or solvent, for example, as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables.

siNAs may also be administered in the form of suppositories, e.g., rectal administration of the drug. These compositions can be prepared by mixing the drug with a non-irritating excipient which is solid at ordinary temperatures and liquid at the rectal temperature and therefore will melt in the rectum to release the drug. These materials include cocoa butter and polyethylene glycols.

siNAs can be extensively modified to enhance stability by modification with nuclease resistant groups, such as 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.57: 163, 1994. The SiNA construct can be purified by gel electrophoresis using a general method or by high performance liquid phase, and then resuspended in water.

Chemically synthesized nucleic acid molecules with modifications (bases, sugars and/or phosphates) prevent their degradation by serum ribonucleases, thus increasing their action. 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 biochem. sd.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 6,300,074. All of the above references describe various chemical modifications that can occur on the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules, significantly enhancing their nuclease stability and efficacy. For example, oligonucleotides are modified with nuclease resistant groups, e.g., 2 ' -amino, 2 ' -C-allyl, 2 ' -fluoro, 2 ' -O-methyl, 2 ' -H nucleotide base modifications to enhance stability and/or enhance biological activity. For a review see Usman and Cedergren, TIBS 17: 34, 1992; usman, et al, Nucleic acids symp.ser.37: 163, 1994; burgin, et al, Biochemistry 35: 14090, 1996. Sugar modifications of nucleotide molecules have been widely described in the art. See Eckstein, et al, international PCT application publication 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.77: 334, 339, 1992; usman, et al, international PCT application publication No. WO 93/15187; sproat, U.S. patent No. 5,334,711 and Beigelman, et al, J biol. chem.270: 25702, 1995; beigelman, et al, international PCT application publication No. WO 97/26270; beigelman, et al, U.S. patent No. 5,716,824; usman, et, U.S. patent No. 5,627,053; woolf, et al, International PCT application publication No. WO 98/13526; thompson, et al, Karpeisky, et al, Tetrahedron lett.39: 1131, 1998; earnshaw and Gait, biopolymers (nucleic Acid sciences) 48: 39-55, 1998; verma and Eckstein, annu.rev.biochem.67: 99-134, 1998; and Burlina, et al, bioorg.med.chem.5: 1999-2010, 1997. These publications describe general methods and strategies for determining the location of sugar, base and/or phosphate modifications, etc., incorporated into nucleic acid molecules that do not modulate catalysis. In view of these teachings, similar modifications described herein can be used to modify siNA nucleic acid molecules of the invention so long as the ability of siNA to promote RNAi in cells is not significantly inhibited.

Although chemical modification of internucleotide linkages of oligonucleotides with phosphorothioate, phosphorodithioate, and/or 5' -methylphosphonate linkages improves stability, excessive modification can result in some toxicity or reduced activity. Thus, the number of these internucleotide linkages should be minimized when designing nucleic acid molecules. Lowering the concentration of these bonds reduces toxicity and results in increased efficacy and higher specificity of these molecules.

In one embodiment, the invention features modified siNA molecules modified with a phosphate backbone comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, carbamate amide, carboxymethyl, imidate, polyamide, sulfonate, sulfonamide, sulfamate, methylal (formacetal), thiomethylal (thioacetal), and/or alkylsilicon (alkylsilyl) substitutions. For a review of oligonucleotide backbone modifications, see huntziker and Leumann, "Nucleic acid analogs: synthesis and Properties, in model Synthetic Methods, "VCH, 1995, pp.331-417, and Mesmaker, et al," Novel Back boron Modifications for Oligonucleotides, in Carbohydrate Modifications in antisense Research, "ACS, 1994, pp.24-39.

In Akhtar, et al, Trends Cell bio.2: 139, 1992; delivery Strategiesfor Antisense Oligonucleotide Therapeutics, ed.akhtar, 1995; maurer, et al, moi.membr.biol.16: 129-140, 1999; hofling and Huang, handb. exp. pharmacol.137: 165-192, 1999; and Lee, et al.. ACS symp.ser.752: 184-192, 2000 describes methods for delivering nucleic acid molecules. Beigelman, et al, U.S. patent nos. 6,395, 713 and Sullivan, et al, PCT publication No. WO 94/02595 further describe general methods of delivery of nucleic acid molecules. These methods can be used for the delivery of essentially any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those skilled in the art, including, but not limited to, encapsulation within liposomes, by iontophoresis, or by incorporation into other carriers, such as biodegradable polymers, hydrogels, cyclodextrins (see, e.g., Gonzalez, et al, Bioconjugate chem.70: 1068-. Alternatively, the nucleic acid/vector composition is delivered locally by direct injection or by use of an infusion pump. Using standard needle and syringe methods or by methods such as those described in Conry, et al, clin. 2330-. The molecules of the present invention may be used as pharmaceutical agents. The pharmaceutical agent prevents, modulates the occurrence of, or treats (alleviates to some extent one symptom, preferably all symptoms) the disease state in the subject.

The term "ligand" refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting, directly or indirectly, with another compound, such as a receptor. The receptor with which the ligand interacts may be present on the cell surface or otherwise be an intracellular receptor. The interaction of a ligand with a receptor may result in a biochemical reaction, or simply a physical interaction or association.

As used herein, "asymmetric hairpin" refers to a linear siNA molecule comprising an antisense region, a loop portion comprising a nucleotide or a non-nucleotide, and a sense region. The sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to pair with the antisense region while forming a double strand loop. For example, an asymmetric hairpin siNA molecule of the invention can include an antisense region (e.g., about 19 to about 22 (e.g., about 19, 20, 21, or 22) nucleotides) and a loop region comprising about 4 to about 8 (e.g., about 4, 5,6, 7, or 8) nucleotides in length sufficient to mediate T cell RNAi, and a sense region comprising about 3 to about 18 (e.g., about 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides complementary to the antisense region. The asymmetric hairpin siNA molecule may also comprise a 5' -phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule may comprise a nucleotide, a non-nucleotide, a linker molecule, or a coupling molecule as described herein.

As used herein, "asymmetric double-stranded" refers to a siNA molecule comprising two separate strands of a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has sufficient complementary nucleotides to pair with the antisense region and form a double strand. For example, an asymmetric double-stranded siNA molecule of the invention can comprise an antisense region (e.g., about 19 to about 22 (e.g., about 19, 20, 21, or 22) nucleotides) of sufficient length to mediate RNAi in T cells and a sense region comprising about 3 to about 18 (e.g., about 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides complementary to the antisense region.

By "modulating gene expression" is meant up-regulating or down-regulating expression of a target gene, including up-regulation or down-regulation of mRNA levels, or mRNA translation, or synthesis of a protein or protein subunit encoded by the target gene, within a cell. 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 target gene being up-or down-regulated, such that the expression, level, or activity of the test protein or subunit is higher or lower than that observed in the absence of the modulator (e.g., siRNA). For example, the term "modulate" may mean "inhibit," but use of the word "modulate" is not limited to this definition.

As used herein, "inhibit," "down-regulate" or "reduce" expression means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the level or activity of one or more proteins encoded by a target gene, is down-regulated to a degree lower than that observed in the absence of a nucleic acid molecule (e.g., siNA) of the invention. In one embodiment, the inhibition, down-regulation or reduction by the siNA molecule is below the level observed in the presence of the inactivating or attenuating molecule. In another embodiment, the inhibition, downregulation, or reduction with siNA molecules is lower than that observed in the presence of scrambled or mismatched siNA molecules. In another embodiment, the suppression, down-regulation or reduction of gene expression with a nucleic acid molecule of the invention is higher than in the absence of the molecule.

"silencing" of a gene refers to the loss of function of part or all of the gene by targeted inhibition of gene expression in a cell and may also be referred to as "knock-down". Depending on the environment and the biological problem to be faced, it may be preferable to partially reduce the gene expression. Alternatively, it may be desirable to reduce gene expression as much as possible. The degree of silencing is determined by methods well known in the art, some of which are reviewed in international PCT application publication No. WO 99/32619. Depending on the assay, quantification of gene expression allows detection of various amounts of inhibition, which is desirable in certain embodiments of the invention, including prophylactic and therapeutic methods, that are capable of knocking down gene expression, e.g., 10%, 30%, 50%, 75%, 90%, 95%, or 99% of gene expression, in terms of mRNA levels or protein levels or activity, that is at or above baseline values (i.e., normal) or that includes elevated expression levels that may be associated with a particular disease state or other state targeted for treatment.

The phrase "inhibiting expression of a target gene" refers to the ability of siNA in the present invention to initiate silencing of the target gene. To detect the degree of gene silencing, a sample or assay of the organism or cultured cells of interest expressing a particular construct is compared to a control sample without construct expression. Control samples (no construct expression) were assigned a relative value of 100%. Inhibition of target gene expression is achieved when the measured value relative to the control is about 90%, often 50%, and in certain embodiments 25-0%. Suitable assays include, for example, protein assays or mRNA level assays using techniques known to those skilled in the art such as dot blotting, northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

As used herein, "subject" refers to an organism, tissue or cell, including a cell of a subject that is a donor or recipient of the subject's organism or transplanted cells or is itself a siNA-delivered subject. Thus, a "subject" can refer to an organism, organ, tissue, or cell, including ex vivo or in vitro treated organ, tissue, or cell subjects for in vivo (ex vivo), to which a nucleic acid molecule of the invention can be administered and enhanced by a polynucleic acid delivery enhancing polypeptide described herein. Exemplary subjects include mammalian individuals or cells, such as human patients or cells.

"cell" as used herein is not meant to refer to an intact multicellular organism, e.g., particularly a human, in its ordinary biological sense. The cell may be present in an organism, for example, birds, plants, and mammals such as humans, cows, sheep, apes, monkeys, pigs, dogs, and cats. The cell may be prokaryotic (e.g., a bacterial cell) or eukaryotic (e.g., a mammalian or plant cell). The cell may be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell may also originate from or comprise a partner or an embryo, stem cell or fully differentiated cell.

By "vector" is meant any nucleic acid-and/or virus-based technique for delivering a nucleic acid of interest.

The use of "including" means including, but not limited to, "including" any content that follows the word. Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional, may or may not be present. The use of "consisting of" means including and limited to any of the phrases "consisting of. Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements are present. The use of "consisting essentially of" is meant to include any elements listed in that phrase, and is not limited to other elements that do not interfere with or contribute to the activity or function of the listed elements as specified in the disclosure. Thus, the phrase "consisting essentially of.. indicates that the listed elements are required or mandatory, but that other elements are optional depending on whether they affect the activity or function of the listed elements, and may or may not be present.

By "RNA" is meant a molecule comprising at least one ribonucleotide group. "ribonucleotide" as used herein refers to a nucleotide bearing a hydroxyl group at the 2' position of the β -D-core-furanose moiety. These terms include double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, substantially purified RNA, synthetic RNA, recombinantly produced RNA, and also include altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. These changes may include the addition of non-nucleotide species to the end or into, for example, siNA, for example at one or more nucleotide positions of the RNA. Nucleotides within RNA molecules of the invention also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNAs.

By "highly conserved sequence region" is meant that the nucleotide sequence of one or more regions of the target gene does not change significantly from one generation to another or from one biological system to another.

The "sense region" is used to refer to a nucleotide sequence in a siNA molecule having a sequence complementary to the antisense region of the siNA molecule. In addition, the sense region of the siNA molecule can comprise a nucleic acid sequence that is homologous to the target nucleic acid sequence.

The term "antisense region" refers to a nucleotide sequence in a siNA molecule having a sequence complementary to a target nucleic acid sequence. In addition, the antisense region of the siNA molecule optionally comprises a nucleotide sequence of a sequence complementary to the sense region of the siNA molecule.

By "target nucleic acid" is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid may be DNA or RNA.

By "complementary" is meant that a nucleic acid is capable of forming hydrogen bonds with another nucleic acid sequence, either through conventional Watson-Crick or other unconventional forms. With reference to the nucleic acid molecules of the present invention, the free energy of binding of a nucleic acid molecule to its complementary sequence is sufficient to allow the relevant function performed by the nucleic acid molecule, e.g., RNAi activity. Determination of the binding free energy of a nucleic acid molecule is well known in the art (see, e.g., Turner, et al., CSHSymp. Quant. biol., LII, 1987, pp. 123-133; Frier, et al., Proc. nat. Acad. Sd. USA 55: 9373. sup. 9377, 1986; Turner, et al., J am. chem. Soc. 709: 3783. sup. 3785, 1987.) the percent complementarity refers to the percentage of adjacent residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with another nucleic acid sequence (e.g., 5,6, 7, 8, 9, and 10 nucleotides out of a total of 10 nucleotides of the first oligonucleotide pair with another nucleic acid molecule having 10 nucleotides, representing 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). "optimally complementary" means that all adjacent residues of a nucleic acid sequence are hydrogen bonded to the same number of adjacent residues of another nucleic acid sequence.

The term "universal base" as used herein refers to a nucleotide base analog that forms a base pair with each of the natural DNA/RNA bases with little distinction between the two. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, oxazolecarboxamide, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole known in the art (see, e.g., Loakes, Nucleic Acids Research 29: 2437-2447, 2001).

The term "acyclic nucleotide" as used herein refers to any nucleotide having an acyclic ribose sugar, for example, wherein any of the ribose carbons (C1, C2, C3, C4, or C5) is independent of or unbound to the nucleotide.

The term "biodegradable" as used herein refers to degradation within a biological system, such as enzymatic or chemical degradation.

The term "biologically active molecule" as used herein refers to a compound or molecule that is capable of initiating or modifying a biological response within a system. Non-limiting examples of biologically active siNA molecules, alone or in combination with other molecules contemplated by the present invention, include therapeutically active molecules such as antibodies, cholesterol, hormones, antiviral drugs, peptides, proteins, chemotherapeutic agents, small molecules, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triple strand forming oligonucleotides (triplex forming oligonucleotides), 2, 5-chimeras, sinas, dsRNA, allelic isozymes, aptamers, decoys, and analogs thereof. The bioactive molecules of the present invention also include molecules that modulate the pharmacokinetics and/or pharmacodynamics of other bioactive molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycols, and other polyethers.

The term "phospholipid" as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, the phospholipid may comprise a phosphorus-containing group and a saturated or unsaturated hydrocarbon group, optionally replaced with OH, COOH, oxo, amine or a substituted or unsubstituted aryl group.

The "cap structure" used refers to a chemical modification that has been incorporated into either end of the oligonucleotide (see, e.g., Adamic, et al, U.S. patent No. 5,998,203, incorporated herein by reference). These end modifications protect the nucleic acid molecule from exonuclease degradation and can aid in nucleic acid molecule delivery and/or localization within the cell. The cap structure may have a 5 '-end (5' -cap) or a3 '-end (3' -cap) or both ends. In non-limiting examples, 5' -caps include, but are not limited to, glyceryl, reverse deoxyabasic residues (moieties); 4 ', 5' -methylene nucleotides; 1- (β -D-erythro-furanosyl) nucleotide, 4' -thio nucleotide; a carbocyclic nucleotide; 1, 5-hexitol anhydride nucleotide; an L-nucleotide; an alpha-nucleotide; nucleotides that modify bases; phosphorothioate linkage; threo-pentofuranosyl (threo-pentofuranosyl) nucleotide; acyclic 3 ', 4' -seco nucleotides; acyclic 3, 4-dihydroxybutyl nucleotide; acyclic 3, 5-dihydroxypentyl nucleotides, 3 '-3' -inverted nucleotide moieties; a3 '-3' -inverted abasic moiety; a3 '-2' -inverted nucleotide moiety; a3 '-2' -inverted abasic moiety; 1, 4-butanediol phosphate; 3' -phosphoramidate; hexyl phosphate ester; aminohexyl phosphate; 3' -phosphate ester; 3' -phosphorothioate; a phosphorodithioate ester; or a bridged or unbridged methylphosphonic acid moiety.

Non-limiting examples of 3' -cap structures include, but are not limited to, glyceryl, reverse deoxyabasic residues (moieties); 4 ', 5' -methylene nucleotides; 1- (. beta. -D-erythro-furanosyl) nucleotide; 4' -thio nucleotides, carbocyclic nucleotides; 5' -amino-alkyl phosphate ester; 1, 3-bisamino-2-propyl phosphate; 3-aminopropyl phosphate ester; 6-aminohexyl phosphate; 1, 2-aminododecyl phosphate; hydroxypropyl phosphate; 1, 5-hexitol anhydride nucleotide; an L-nucleotide; an alpha-nucleotide; nucleotides that modify bases; a phosphorodithioate ester; threo-pentofuranosyl (threo-pentofuranosyl) nucleotide; acyclic 3 ', 4' -seco nucleotides; 3, 4-dihydroxybutyl nucleotide; 3, 5-dihydroxypentyl nucleotide, 5 '-5' -inverted nucleotide moiety; a 5 '-5' -inverted abasic moiety; 5' -phosphoramidate; 5' -phosphorothioate; 1, 4-butanediol phosphate; 5' -amino; bridged or unbridged 5 '-phosphoramidates, phosphorothioates and/or phosphorodithioates, bridged or unbridged methylphosphonates and 5' -mercapto moieties (see Beaucage and Lyer, Tetrahedron 49: 1925, 1993 for more details; incorporated herein by reference).

The term "non-nucleotide" as used herein refers to any group or compound that can be incorporated into a nucleic acid strand at one or more nucleic acid units and can allow the remaining bases to exert their enzymatic activity, including sugar and/or phosphate substitutes. The group or compound is abasic in that it does not contain commonly recognized nucleotide bases such as adenine, guanine, cytosine, uracil or thymine, and thus there is no base at the 1' -position.

"nucleotides" as used herein, as is art-recognized, includes both natural bases (standard) and modified bases well known in the art. These bases are typically located at the 1' position of the sugar portion of the nucleotide. Nucleotides generally include base, sugar, and phosphate groups. The nucleotide may be unmodified or modified in the sugar, phosphate, and/or base portion (and may alternatively be referred to as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, and others; see, e.g., Usman and McSwiggen, supra; Eckstein, et al., International PCT application publication No. WO 92/07065; Usman, et al., International PCT application publication No. WO 93/15187; Uhlman & Peyman, supra, all of which are hereby incorporated by reference). Some examples of modified nucleobases known in the art are described by Limbach, et al, Nucleic Acids res.22: 2183, 1994. Some non-limiting examples of base modifications that can be introduced into a nucleic acid molecule include inosine, purine, tetrahydropyridine, dihydropyridine, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyluracil, dihydrouracil, naphthyl, aminophenyl, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., thymine), 5-halouracil (e.g., 5-bromouracil) or 6-azapyrimidine or 6-alkylpyrimidine (e.g., 6-methyluracil), propyne, and others (Burgin, et al., Biochemistry 35: 14090, 1996; Uhlman & Peyman, supra). "modified base" as used in this regard refers to a nucleobase other than adenine, guanine, cytosine and uracil at the 1' position or an equivalent thereof.

By "target" is meant a sequence within the target RNA that is targeted for cleavage mediated by a siNA construct containing a sequence complementary to the target sequence in the antisense region.

By "detectable level of cleavage" is meant that the target RNA is cleaved (and the cleavage product RNAs are formed) to an extent sufficient to distinguish the cleavage products against the background of RNA produced by random degradation of the target RNA. The production of 1-5% of degradation products of the target RNA is sufficient for most detection methods to detect against background.

As used herein, "biological system" refers to a purified or non-purified material from a biological source including, but not limited to, humans, animals, plants, insects, bacteria, viruses, or other sources, wherein the system includes components necessary for RNAi activity. The term "biological system" includes, for example, a cell, tissue, or organism, or an extract thereof. The term biological system also includes recombinant RNAi systems that can be used in an ex vivo environment.

The term "biodegradable linker" as used herein refers to a nucleic acid or non-nucleic acid linker molecule designed as a biodegradable linker that links one molecule to another molecule, e.g., to a siNA molecule of the invention or sense and antisense strands of a siNA molecule of the invention to a biologically active molecule. The biodegradable linker is designed such that its stability can be modulated according to a particular purpose, such as delivery to a particular tissue or cell type. The stability of nucleic acid-based biodegradable linker molecules can be adjusted using various chemical methods, such as a combination of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, such as 2 ' -O-methyl, 2 ' -fluoro, 2 ' -amino, 2 ' -O-amino, 2 ' -C-allyl, 2 ' -O-allyl, and other 2 ' -modified or base-modified nucleotides. The biodegradable nucleic acid linking molecule can be a dimer, trimer, tetramer, or longer nucleic acid molecule, e.g., 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 containing a single nucleotide based on a phosphorus linkage, e.g., a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also include modifications of the nucleic acid backbone, nucleic acid sugars, or nucleic acid bases.

"abasic" as used herein refers to sugar moieties that lack a base or have other chemical groups at the 1' base position, see, e.g., Adamic, et al, U.S. Pat. No. 5,998,203.

The term "unmodified nucleotide" as used herein refers to one of the bases adenine, cytosine, guanine, thymine or uracil attached to the 1' carbon position of a β -D-ribofuranose.

By "modified nucleoside" is meant any nucleotide base that contains a modification at the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Formulas I-VII and/or other modifications described herein show non-limiting examples of modified nucleotides.

In connection with the 2 '-modified nucleotides described in the present invention, the "amino" used means 2' -NH₂Or 2' -O-NH₂It may be modified or unmodified. These modified groups are described, for example, in Eckstein et al, U.S. Pat. No. 5,672,695 and Maralic-Adamic, et al, U.S. Pat. No. 6,248,878.

siNA molecules can be complexed with cationic lipids, encapsulated within liposomes, or otherwise delivered to a target cell or tissue. The nucleic acid or nucleic acid complex may be combined or not combined with the biopolymer and administered locally by injection, infusion pump or stent. In another embodiment, polyethylene glycol (PEG) can be covalently bound to the siNA compounds, polynucleotide delivery-enhancing polypeptides, or both of the present invention. The conjugated PEG may be of any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).

The sense region may be linked to the antisense region by a linker molecule such as a polynucleotide linker or a non-nucleotide linker.

"inverted repeat" refers to a nucleic acid sequence comprising sense and antisense elements positioned such that when the repeat sequence is transcribed, they form a double stranded siRNA. The inverted repeat may optionally include a linker or a heterologous sequence such as a self-cleaving ribozyme between the two elements of the repeat. The inverted repeat element is of sufficient length to form a double stranded RNA. Typically, each element of the inverted repeat is about 15 to about 100 nucleotides in length, preferably about 20-30 base nucleotides in length, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

"nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which may be synthetic, naturally occurring, and non-naturally occurring, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

"Large double-stranded RNA" refers to any double-stranded RNA having a length greater than 40 base pairs (bp), e.g., greater than 100bp or more particularly greater than 300 bp. The sequence of the large double strand may be a fragment of an mRNA or an entire mRNA. The maximum size of the large dsRNA is not limited in the present invention. The double stranded RNA may comprise a modified base, wherein the modification may be a modification of the phosphate sugar backbone or a nucleoside. These modifications may include nitrogen, sulfur heteroatoms, or any other modification known in the art.

The double-stranded structure may be formed by the annealing of self-complementary RNA strands or two different complementary RNA strands present in, for example, a hairpin structure or microRNA.

"overlap" refers to a sequence in which two RNA fragments have multiple nucleotides overlapping on one strand, e.g., where the number of the multiple nucleotides is as little as 2-5 nucleotides or about 5-10 nucleotides or more.

"one or more dsRNAs" refers to dsRNAs that are different from each other in base sequence.

"target gene or mRNA" refers to any gene or mRNA of interest. In fact, any of the genes previously identified by genetics or sequencing represents a target. Target genes or mrnas include developmental genes and regulatory genes in addition to metabolic or structural genes or genes encoding enzymes. The target gene may be expressed in the cell whose phenotype is being studied or in an organism in a manner that directly or indirectly affects the phenotypic characteristics. The target gene may be endogenous or exogenous. These cells include any cell within the body of an adult or embryonic animal or plant including gametes, or any isolated cell present in an immortalized cell line or primary cell culture.

In this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Examples

The foregoing disclosure generally describes the present invention, which is further illustrated by the following examples. These examples are described only to illustrate the present invention and do not limit the scope of the present invention. Although specific terms and values are employed herein, they are to be understood as exemplary and not limiting the scope of the invention.

Example 1

Compositions comprising siRNA complexed with a polynucleotide delivery-enhancing polypeptide Preparation and characterization of

To form a complex between candidate siRNAs of the present invention and a polynucleotide delivery-enhancing polypeptide, an appropriate amount of siRNA is combined with a predetermined amount of polynucleotide delivery-enhancing polypeptide, e.g., as inIn cell culture medium (Invitrogen), at the indicated ratios, and incubated at room temperature for about 10-30 minutes. Subsequently, a selected volume, for example, about 50. mu.l of the mixture is contacted with the target cells, and the cells are incubated for a predetermined incubation time, in this example about 2 hours. The siRNA/peptide mixture may optionally include cell culture media or other additives such as fetal bovine serum. A series of experiments were performed on histones H3, H4 and H2bExperiments combined these polynucleotide delivery-enhancing polypeptides with siRNA in varying ratios. Generally, the siRNA/histone ratio starts at 1: 0.01 to 1: 50. 40pm siRNA was added per well in a 96-well microplate. Each well contained a 50% confluent monolayer of β -gal cells. An exemplary optimized ratio of transfection efficiencies is shown in table 2 below.

Transfection was performed on 9L/. beta. -gal cells with conventional siRNA or siRNA complexed with one of the above-identified histones. The siRNA was designed to specifically knock down β -galactosidase mRNA, activity expressed as a percentage of β -gal activity of control (control cells transfected with liposomes without polynucleotide delivery enhancing polypeptide).

Assays to detect and/or quantify the efficiency of siRNA delivery are performed using conventional methods, such as β -galactosidase assays or flow cytometry methods.

Beta-galactosidase assays were performed using 9L/LacZ cells, a cell line constitutively expressing beta-galactosidase. 9L/LacZ cells are rat keratinocyte fibroblast cells constitutively expressing LacZ, obtained from ATCC (# CRL-2200). 9L/LacZ cells were grown in Dulbecco's Modified Essential Medium (DMEM) supplemented with 1mM sodium pyruvate, non-essential amino acids and 20% fetal bovine serum. Cells were incubated at 37 ℃ with 5% CO₂Cultured and supplemented with a mixture of antibiotics containing 100 units/ml penicillin, 100. mu.g/ml streptomycin and 0.25mg/ml amphotericin B (Invitrogen). siRNA duplexes designed against β -gal mRNA were chemically synthesized and used with delivery reagents to assess knockdown efficiency.

Peptide synthesis

Peptides were synthesized by solid phase Fmoc chemistry on CLEAR-amide resin using a Rainin Symphony synthesizer. The coupling step was performed with 5 equivalents of HCTU and Fmoc amino acids in excess of N-methylmorpholine for 40 min. The peptide resin was treated twice with 20% pyridine in DMF for 10 min each to remove Fmoc. Upon completion of the entire peptide synthesis, the Fmoc group was removed with pyridine and washed extensively with DMF. The maleimide-modified peptide was prepared by coupling 3.0 equivalents of 3-maleimidopropionic acid and HCTU to the N-terminus of the peptide resin in the presence of 6 equivalents of N-methylmorpholine. The extent of coupling was monitored by the Kaiser test. The peptide was cleaved from the resin by the addition of 10mL of TFA containing 2.5% water and 2.5 triisopropylsilane (trisisopropylsilane) followed by gentle stirring at room temperature for 2 hours. The crude peptide produced was collected by trituration with ether followed by filtration. The crude product was dissolved in Millipore water and lyophilized. The crude peptide was dissolved in 15mL of water containing 0.05% TFA and 3mL of acetic acid, and loaded onto a Zorbax RX-C8 reverse phase column (22mm ID. times.250 mm, 5 μm particle size) through a 5mL injection loop at a flow rate of 5 mL/min. Purification was accomplished by performing a linear AB gradient elution of 0.1% B/min, where solvent a was 0.05% TFA in water and solvent B was 0.05% TFA in acetonitrile. The purified peptide was analyzed by HPLC and ESMS.

siRNA synthesis and preparation

The synthesis of oligonucleotides was accomplished using standard 2-cyanoethylphosphonite procedures on a long chain alkylamine controlled pore glass derived from either a selection of 5 ' -O-dimethyltrityl-2 ' -O-t-butyldimethylsilane ribonucleotides or the applicable 5 ' -O-dimethyltrityl-2 ' -deoxy-3 ' -O-succinylthymine vector. All oligonucleotides were synthesized on a 0.2 or 1- μmol scale using an ABI 3400DNA/RNA synthesizer, using concentrated NH₄OH is cleaved from the solid support and NH is mixed 3: 1₄EtOH deprotection at 55 ℃. Deprotection of the 2' -TBDMS protecting group was achieved by incubating the base-deprotected RNA with a solution of N-methylpyrrolidone/triethylamine tris (fluorohydric acid) (6: 3: 4 by volume) (600. mu.L per. mu. mol) at 65 ℃ for 2.5 hours. The corresponding synthesis module: A. u, C and G of 5 ' -dimethoxytrityl-N- (tac) - ' -O- (t-butyldimethylsilane) -3 ' - [ (2-cyanoethyl) - (N, N-diisopropyl)]Phosphoramidite (Prologo, Boulder CO) and 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) was purchased directly from commercial suppliers. Triethylamine-trihydrofluoride, N-methylpyrrolidone and concentrated ammonium hydroxide were purchased from Aldrich. All HPLC analyses and purifications were carried out with Xterra^TMWaters 2690 on the column. All other reagents were purchased from Glen Research Inc. The oligonucleotides were purified to over 97% purity as determined by RP-HPLC. siRNAs for injection into mice were purchased from Qiagen, annealed and purified by HPLC, containing acceptable endotoxin levels for in vivo injection.

Cell culture

Primary human monocytes

Fresh human blood samples from healthy donors were purchased from Golden West Biologicals. To isolate the monocytes, blood samples were received and diluted 1: 1 with PBS. Peripheral Blood Mononuclear Cells (PBMC) were first separated from whole blood by ficoll (amersham) gradient. Monocytes were further purified from PBMCs using the Miltenyi CD14 positive selection kit and protocol provided (milltenyi BIOTEC). To assess the purity of the monocyte preparations, cells were incubated with anti-CD 14 antibody (BD Biosciences) and then sorted by flow cytometry. Purity of the monocyte preparation was higher than 95%.

The procedure for activating human monocytes was performed by adding 0.1-1.0ng/ml lipopolysaccharide LPS (Sigma, St Louis, Mo.) to cultured cells to stimulate TNF- +/-production. Cells were harvested after 3 hours incubation with LPS and mRNA levels were determined by Quantigene assay (genospecra, Fremont, CA) according to the manufacturer's instructions.

Mouse tail fibroblast

Mouse Tail Fibroblast (MTF) cells were derived from the tail of C57BL/6J mice. The tails were cleaved and immersed in 70% ethanol and then cut into small pieces with a razor blade. These fragments were washed three times with PBS and then incubated with 0.5mg/mL collagenase, 100 units/mL penicillin and 100. mu.g/mL streptomycin on a 37 ℃ shaker to disruptBad tissue. The tail fragment was then cultured in complete medium (Dulbecco's modified essential medium containing 20% FBS, 1mM sodium pyruvate, non-essential amino acids and 100 units/mL penicillin and 100. mu.g/mL streptomycin) until cells were established. Cells were incubated at 37 ℃ with 5% CO₂Next, the culture was carried out in the complete medium as listed above.

Transfection procedure

The first day of the procedure, saturated 9L/LacZ cultures were obtained from T75 flasks, and the cells were then isolated and diluted into 10ml of complete medium (DMEM, 1xPS, 1 Xsodium pyruvate, 1 XNEAA). The cells were further diluted 1: 15 and then 100. mu.l of this preparation was aliquoted into the wells of a 96-well plate, which would yield about 50% cell confluence the next day of transfection. The edge wells were left empty, filled with 250. mu.l of water, and the plates were then placed in an incubator overnight (5% CO) without stacking at 37 ℃₂An incubator).

The next day, 50. mu.l of transfection complex per well was prepared in Opti-MEM. The medium was removed from the plate and the wells were washed once with 200. mu.l PBS or Opti-MEM. By inversion, the plates were completely blotted dry. The transfection mix was then added (50. mu.l/well) to each well and 250. mu.l of water was added to the marginal wells to prevent drying. Cells were then incubated at 37 deg.C (5% CO)₂Incubator) for at least 3 hours. The transfection mixture was removed and replaced with 100. mu.l of complete medium (DMEM, 1XPS, 1 XPEAA). The cells were cultured for a defined length of time and then harvested for enzymatic analysis.

Cell viability (MTT assay)

Cell activity was assessed using the MTT assay (MTT-100, MatTek kit). The kit measures the uptake of tetrazolium salt and the conversion of tetrazolium salt to formazan dye. Thawed and diluted MTT concentrates were prepared by mixing 2mL of MTT concentrate with 8mL of MTT diluent 1 hour prior to the end of the lipid administration period. Ca-containing per cell culture insert (insert)⁺²And Mg⁺²Washed twice with PBS, and then transferred to a well containing 100. mu.L of mixMTT solution in a new 96-well transport plate. The 96-well transfer plate was incubated at 37 ℃ with 5% CO₂And incubated for 3 hours. After 3 hours of incubation, the MTT solution was removed and the culture was transferred to another 96-well feeder tray containing 250 μ L of MTT extract per well. An additional 150. mu.L of MTT extract was added to the surface of each culture well and the samples were placed in the dark at room temperature for a minimum of 2 hours and a maximum of 24 hours. The insert membrane is then pierced with a tip to mix the liquids in the upper and lower wells. 200 microliters of this combined extract and extraction blank (negative control) were transferred into a 96-well plate and measured using a microplate reader. The Optical Density (OD) of the sample was measured at 570nm of the plate reader, with background subtraction at 650 nm. Cell viability was expressed as a percentage and was calculated by dividing the OD reading of the insert in the treated group by the OD reading of the insert treated with PBS and multiplying by 100. For the purposes of this analysis, PBS is assumed to have no effect on cell viability and therefore represents 100% cell viability.

Enzyme assay

Reagents for enzyme detection were purchased from Invitrogen (β -Gal assay kit) and Fisher (Pierce micro BCA protein assay kit, Catalog)

A: cell lysis

Remove the medium, wash once with 200 μ l PBS, invert the blotter plate.

Add 30. mu.l of lysate from the beta-Gal kit per well.

Freeze-thaw the cells twice to produce a lysate.

B: beta-Gal analysis

The assay mix (50. mu.l 1 Xbuffer, 17. mu.l ONPG per well) was prepared.

Fresh plates were removed and 65. mu.l of assay mix was added to each well.

Add 10. mu.l of cell lysate per well. Blank wells should be prepared to remove background activity.

Long incubation periods with about 20 min at 37 ℃ to prevent depletion of ONPG, favoring high expression.

Add 100. mu.l stop buffer.

OD was measured at 420 nm.

C: BCA assay

BSA standards (150 ul per well) were prepared and duplicate spots were applied to each plate.

Add 145. mu.l of water to each well and 5. mu.l of cell lysate to each well.

The final assay reagent was prepared according to the manufacturer's instructions.

Add 150. mu.l of detection reagent to each well.

Incubate at 37 ℃ for about 20 minutes.

OD was measured at 562 nm.

D: calculation of specific Activity

Specific activity expressed as nmol hydrolysed ONPG/t/mg protein, where t is the minute time of incubation at 37 ℃; mg protein is the test protein determined by the BCA method.

Flow cytometry assay of FITC/FAM conjugated siRNA

Fluorescence activated cell sorting analysis was performed using a Beckman Coulter FC500 cell analyzer (Fullerton, CA). The instrument was adjusted according to the fluorescent probes used (FAM or Cy5 labeled with siRNA and FITC and PE labeled with CD 14). Propidium iodide (Fluka, St Lois, MO) and AnnexinV (R & D system, Minneapolis, MN) were used as indicators of cell viability and cytotoxicity. A brief protocol of the steps is detailed below.

a) After exposure to the siRNA/peptide complex, the cells were incubated for at least 3 hours.

b) Cells were washed with 200. mu.l PBS.

c) Cells were detached with 15. mu.l TE and incubated at 37 ℃.

d) Cells in 5 wells were resuspended with 30 μ l FACS solution (PBS containing 0.5% BSA and 0.1% sodium azide).

e) All 5 well cells were pooled into 1 tube.

f) Mu.l of PI (propidium iodide) were added to each tube.

g) Cells were analyzed by fluorescence activated cell sorting (FCAS) according to the manufacturer's instructions.

The siRNA sequence for silencing β -galactosidase mRNA is as follows:

C.U.A.C.A.C.A.A.A.U.C.A.G.G.A.U.U.U.DT.DT (sense) (SEQ ID NO: 32)

A.A.U.C.G.C.U.G.A.U.U.U.G.U.G.U.U.A.G.dT.dT (antisense) (SEQ ID NO: 33)

Table 2 shows the data of this example. The transfection efficiency is inversely proportional to the amount of beta-galactosidase activity measured in the cell lysate. Upon transfection, a decrease in the activity of the measured beta-galactosidase is indicative of successful transfection. Thus, in the absence of transfection, the activity of the β -galactosidase enzyme measured was 100% and the transfection efficiency was O%. Transfection efficiency increases as the activity of beta-galactosidase decreases. For example, in table 2, histone H2B with siRNA resulted in a transfection efficiency of 62.03%, indicating a 37.97% reduction in the activity of the measured β -galactosidase. The data presented in Table 3 were obtained using the same method for determining transfection efficiency.

Table 2:

enhancement of polypeptide-mediated siRNA delivery efficiency by polynucleotide delivery in 9L/LacZ cells

Transfection mixture	Transfection efficiency (% Total cells)	The molar ratio is as follows: (siRNA: peptide)
			SiRNA alone (40 pmol/well)	0.09％
Cationic lipids (Invitrogen)	84.32％	unknown
			Histone H2B	62.03％	1∶10-15
Histone H3	85.08％	1∶10-20
			Histone H4	72.07％	1∶4-8
GEQIAQLIAGYIDIILKKKKSK(SEQ ID NO：31)	50.86％	1∶5-20
			WWETWKPFQCRICMRNFSTRQARRNHRRRHR(SEQ ID NO：27)	98.29％	1∶0.5-4
Poly Lys-Trp, 4: 1, MW20,000-	71.92％	1∶2-8
			Poly Orn-Trp, 4: 1, MW20,000-	74.16％	1∶2-8

siRNA/peptide/lipid

To evaluate the effect of adding cationic lipids to the siRNA/polynucleotide delivery-enhancing polypeptide mixture, complex or conjugate, the above procedure was repeated except that liposomes (Invitrogen) were added to the siRNA/polynucleotide delivery-enhancing polypeptide formulation at a constant concentration according to the manufacturer's instructions.

For preparation of a peptide consisting of GKINLKALAALAKKIL (SEQ ID NO: 28), siRNA and(Invitrogen), siRNA and peptide were first mixed together in Opti-MEM medium at room temperature, after which they were mixed together at room temperatureAdded to the mixture to form a siRNA/peptide/cationic lipid composition.

For preparation of a peptide consisting of RVIRVWFQNKRCKDKK (SEQ ID NO: 29), siRNA andcompositions of matter, peptides andfirst mixed together in Opti-MEM cell culture medium, siRNA was added to the mixture to form siRNA/peptideA composition is provided.

For the preparation of siRNA/peptide/cationic lipid compositions using GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30) or GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), it is not important in what order these ingredients are added together to prepare the siRNA/peptide/cationic lipid composition.

For the preparation of siRNA/melittin-siRNA and melittin were first mixed together in Opti-MEM cell culture medium and thenIs added to the mixture.

To prepare siRNA/HistoneComposition, Histone H1 andfirst added together to the Opti-MEM cell culture medium, mixed well, then siRNA was added, with histoneThe mixture was mixed well to form siRNA/histone H1A composition is provided.

Table 3:

polypeptide-mediated siRNA delivery efficiency by polynucleotide delivery enhancement in 9L/LacZ cells with or without cationic lipids

Transfection mixture	Transfection efficiency with lipids (% total cells)	Transfection efficiency without lipids (% total cells)	siRNA to peptide ratio in transfection mixtures
				SiRNA alone	1.72％	0.11％
Liposome (without peptide)	83.48％
				GKINLKALAALAKKIL(SEQ ID NO：28)	89.67％	0.26％	1∶5-20
RVIRVWFQNKRCKDKK(SEQ ID NO：29)	89％	0.59％	1∶1-5
				GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ(SEQ ID NO：30)	89.99％	54.58％	1∶5
GEQIAQLIAGYIDIILKKKKSK(SEQ ID NO：31)	90.01％	50.86％	1∶5-10
				Melittin	93.1％	5.15％	1∶20
Histone H1	93.39％	0.14％	1∶10-20

Based on the foregoing results, exemplary polynucleotide delivery-enhancing polypeptides of the invention substantially enhance cellular uptake of siRNAs, while the addition of optional cationic lipids to certain siRNA/polynucleotide delivery-enhancing polypeptide mixtures of the invention substantially increases siRNA delivery efficiency.

Example 2

Preparation of a composition comprising siRNA conjugated to a TAT-HA polynucleotide delivery-enhancing polypeptide And features of

This example describes the synthesis and uptake activity of a specific peptide covalently coupled to one of the siRNA duplexes. These conjugates provide efficient delivery of siRNA to the cytosol.

Peptide synthesis

Peptides were synthesized by solid phase Fmoc chemistry on CLEAR-amide resin using a Rainin Symphony synthesizer. The coupling step was performed with 5 equivalents of HCTU and Fmoc amino acids in excess of N-methylmorpholine for 40 min. The peptide resin was treated twice with 20% pyridine in DMF for 10 min each to remove Fmoc. Upon completion of the synthesis of the whole peptide, the Fmoc group was removed with pyridine and washed thoroughly with DMF. The maleimide-modified peptide was prepared by coupling 3.0 equivalents of 3-maleimidopropionic acid and HCTU to the N-terminus of the peptide resin in the presence of 6 equivalents of N-methylmorpholine. The extent of coupling was monitored by the Kaiser test. The peptide was cleaved from the resin by the addition of 10mL TFA containing 2.5% water and 2.5 triisopropylsilane followed by gentle stirring at room temperature for 2 hours. The crude peptide produced was collected by trituration with ether followed by filtration. The crude product was dissolved in Millipore water and lyophilized. The crude peptide was dissolved in 15mL of water containing 0.05% TFA and 3mL of acetic acid, and loaded onto a Zorbax RX-C8 reverse phase column (22mm ID. times.250 mm, 5 μm particle size) through a 5mL injection loop at a flow rate of 5 mL/min. Purification was accomplished by running a linear AB gradient of 0.1% B/min, where solvent a was 0.05% TFA in water and solvent B was 0.05% TFA in acetonitrile. The purified peptides were analyzed by HPLC and ESMS.

Synthesis of conjugates

Peptides and RNAs were prepared using standard solid phase synthesis methods. The peptide and siRNA molecule must incorporate functional groups with specific moieties to allow covalent binding to each other. For peptides, the N-terminus is functionalized, for example, with 3-maleimidopropionic acid. However, other functional groups such as bromo or iodoacetoxy are known to work as well. For RNA molecules, either the 5 'end of the sense strand or the 3' end of the antisense strand is functionalized with, for example, a 1-O-dimethoxytrityl-hexyl-disulfide linker, according to the following synthetic method.

The 5' modified C6 SS-oligonucleotide (GCAAGCUGACCCUGAAGUUCAU (SEQ ID NO: 34); 3.467 mg; 0.4582. mu. mol) was reacted with 0.393mg (3eq) of tris (2-carboxyethyl) phosphine (TCEP) in 0.3ml of 0.1M triethylamine acetate (TEAA) buffer (pH 7.0) at room temperature for 3h to reduce the free thiol group. The reduced oligonucleotide was purified by RP HPLC inMS C₁₈4.6X 50mm column with a linear gradient of 0 to 30% CH in 0.1M TEAA buffer (pH 7)₃CN Wash column for 20 min (t)_r＝5.931min)

The purified reduced oligonucleotide (1.361mg, 0.19085 μ tnol) was dissolved in 0.2ml of 0.1M TEAA buffer (pH 7), and then the peptide having a maleimide moiety bound to the N-terminus of the peptide (0.79mg, 1.5eq) was added to the oligonucleotide solution. Upon addition of the peptide, a precipitate formed immediately, which was formed upon addition of 150. mu.l of 75% CH₃CN/0.1M TEAA disappeared. After stirring overnight at room temperature, the resulting conjugate was purified by RP HPLC atMS C184.6X 50mm column with a linear gradient of CH from 0 to 30% in 0.1M TEAA buffer (pH 7)₃The column was washed with CN for 20 minutes, followed by 100% C for the next 5 minutes. The amount of conjugate was determined spectrophotometrically, based on the molar absorption coefficient calculated at λ -260 nm. MALDI mass spectrometry analysis revealed that the peak of the observed conjugate (10585.3amu) matched the calculated mass. Yield: 0.509mg, 0.04815. mu. mol, 25.2%.

The peptide conjugate sense strand and the appended antisense strand were annealed in 50mM potassium acetate, 1mM magnesium acetate and 15mM HEPES, pH 7.4 by heating at 90 ℃ for 2 minutes followed by incubation at 37 ℃ for 1 hour. The formation of double stranded RNA conjugates was confirmed by non-denaturing (15%) polyacrylamide gel electrophoresis followed by ethidium bromide staining.

Structure of peptide-siRNA conjugates (SEQ ID NOS 34 and 35)

Uptake assay

Cells were plated in 24-well plates one day prior to transfection to allow 50-80% confluent monolayers at the time of transfection. For complex formation, siRNA and peptide were used in Opti-Media (Invitrogen) was diluted and then mixed and allowed to complex for 5-10 minutes before adding to the cells washed with PBS. At each peptide concentration (2-50. mu.M), the final concentration of siRNA was 500 nM. The conjugate was also diluted in Opti-The final concentration added to the cells in the medium ranged from 62.5nM to 500 nM. At a concentration of 500nM, we also combined the conjugate with 20% FBS just before it was added to the cells. At 37 ℃ 5% CO₂Cells were transfected for 3 hours under conditions. Cells were washed with PBS, treated with pancreatin, and then analyzed by flow cytometry. siRNA uptake was measured by Cy5 fluorescence intensity and cell viability was assessed by addition of propidium iodide.

As shown in figure 1, there was a higher percentage uptake of peptide/siRNA conjugate in mouse tail fibroblasts compared to peptide/siRNA complexes. Further, the peptide/siRNA conjugates had higher mean fluorescence intensity (MFI; FIG. 2) compared to the peptide/siRNA complexes. Thus, these data suggest that in certain embodiments it may be desirable to conjugate a polynucleotide delivery-enhancing polypeptide to an siRNA molecule.

Practice ofExample 3

screening of siRNA/delivery peptide complexes demonstrates rationally designed polynucleotide delivery of diverse assemblies Delivery of enhancing polypeptide effectively induces siRNA uptake in 9L/LacZ cells

This example provides additional evidence that the rationally designed polynucleotide delivery-enhancing polypeptides of the invention, when complexed to siRNAs, enhance siRNA uptake in a widely diverse assembly.

In flat bottom 96-well plates, approximately 10,000 9L/lacZ cells were plated per well, allowing approximately 50% confluence to be reached at the next day of transfection. FAM-labeled siRNA and peptides in Opti-Diluted 2-fold to final concentration in medium (Invitrogen). The siRNA and peptide were mixed in equal volumes, complexed for 5-10 minutes at room temperature, and 50. mu.L was added to cells previously washed with PBS. 5% CO at 37 ℃₂Cells were transfected for 3 hours under conditions. Cells were washed with PBS, treated with pancreatin, and then analyzed by flow cytometry. siRNA uptake was measured by FAM fluorescence intensity and cell viability was assessed by addition of propidium iodide. Table 4 below summarizes the data on the percentage of cellular uptake of the polypeptides in 9L/LacZ cells corresponding to various rationally designed polynucleotide delivery-enhancing polypeptides. The concentrations of the peptides and siRNA used are included in table 4.

Table 4:

in 9L/LacZ cells, rational design of polynucleotide delivery enhanced polypeptide mediated siRNA uptake efficiency

Example 4

Ex vivo siRNA/delivery enhanced by polynucleotide delivery enhancing polypeptide

This example illustrates that siRNA uptake is enhanced by a polynucleotide delivery-enhancing polypeptide of the invention in LacZ cells, murine primary fibroblasts, and human monocytes. Experiments performed on 9L/LacZ cells and mouse fibroblasts generally used the same materials and methods as described above, except that Mouse Tail Fibroblasts (MTF) were substituted for 9L/LacZ cells in the murine experiments. The materials and methods used for experiments performed on human monocytes are described later. Table 5 summarizes the results of transfection on MTF cells. Table 5 includes the amino acid sequences of the peptides used, as well as the peptide and concentration of Cy5 label coupled to eGFP siRNA. Table 6 summarizes the results of transfection on MTF and 9L/LacZ cells. The data presented in table 6 provides a comparison of the transfection efficiency of some peptide/siRNA complexes in different cell types.

Table 5:

in rat tail fibroblast (MTF) cells, the efficiency of siRNA uptake mediated by rationally designed polynucleotide delivery enhancing polypeptides

Peptide ID #	Amino acid sequence	Status of state	% intake
				PN250	NH 2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-amide (SEQ ID NO: 35)	0.5mMsiRNA/40mM peptide	85.9％
PN73	NH 2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 59)	0.5mM siRNA/5mM peptide	94.5％
				PEG-PN509	Peg-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 90)	0.5mMsiRNA/25mM peptide	91％
PN404	NH 2-RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ-amide (SEQ ID NO: 91)	0.5mMsiRNA/25mM peptide	50.4％
				PN361	NH 2-KKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 58)	0.5mMsiRNA/50mM peptide	65％
PN27	AAVALLPAVLLALLAPRKKRRQRRRPPQC(SEQ ID NO：38)	0.5mM siRNA/5mM peptide	60.7％
				PN58	NH 2-RQIKIWFQNRRMKWKK-amide (SEQ ID NO: 53)	1mM siRNA/20mM peptide	3.7％
PN158	NH2-RVIRWFQNKRCKDKK amide (SEQ ID NO: 67)	0.5mMsiRNA/50nM peptide	86.2％
				PN316	Maleimido-RVIRWFQNKRSKDKK-amide (SEQ ID NO: 92)	0.5mMsiRNA/100mM peptide	84.8％
PN289	Maleimide-WRFKQqqqqqqqqqqqq-amide (SEQ ID NO: 76)	0.5mMsiRNA/10mM peptide	7％
				PN28	NH 2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-amide (SEQ ID NO: 39)	1mM siRNA/8mM peptide	80.5％
PN173	GRKKRRQRRRPPQC(SEQ ID NO：36)	0.5mMsiRNA/130nM peptide	94.8％
				PN159	KLALKLALKALKAALKLA-amide (SEQ ID NO: 13)	0.5mM siRNA/5mM peptide	0％
PN161	NH 2-GWTLNSAGYLLGKINLKALAALAKKIL-amide (SEQ ID NO: 93)	0.5mMsiRNA/10nM peptide	0％

Table 6:

polypeptide-mediated efficiency of siRNA uptake by rationally designed polynucleotide delivery in LacZ cells and rat tail fibroblasts

To further characterize polynucleotide delivery enhancing the ability of polypeptides to transfect cultured cells, human monocytes were incubated with 200nM FITC-labeled siRNA complexed with different concentrations of PN73, PN250, PN182, PN58, and PN 158. In addition to LacZ and murine fibroblasts, human monocytes were used because they are the targeted cell type in the treatment of rheumatoid arthritis.

Fresh human blood samples from healthy donors were purchased from Golden West Biologicals. To isolate the monocytes, blood samples were immediately diluted 1: 1 with PBS upon receipt. Peripheral Blood Mononuclear Cells (PBMCs) were first isolated from whole blood by ficoll (amersham) gradient method. Monocytes were further purified from PBMCs using the Miltenyi CD14 positive selection kit and the protocol provided (millenyi BIOTEC). To assess the purity of the monocyte preparations, cells were incubated with anti-CD 14 antibody (BD Biosciences) and then sorted by flow cytometry. Purity of the monocyte preparation was higher than 95%.

The following description is a brief summary of the transfection protocol used in this example. Plating is performed when adherent cells reach 70-90% confluent monolayer and suspension cells have 100,000 cells per well. To prepare siRNA/transfection reagent complexes, Cy 5-or FAM-coupled siRNA and peptide, respectively, were prepared in Opti-Diluted 2-fold to final concentration in medium (Invitrogen). The siRNA and transfection reagent are mixed in equal volumes and complexed for 5-10 minutes at room temperature. For siRNA-peptide conjugates, the conjugates were directly in Opti-Dilution in culture medium. The transfection mixture was added to cells previously washed with PBS. At 37 deg.C, 5% CO₂Cells were transfected for 3 hours. To analyze siRNA uptake, cells were washed with PBS, treated with pancreatin (only for adherent cells), and then analyzed by flow cytometry. siRNA uptake was determined by intracellular Cy5 or FAM fluorescence intensity. Cell viability was determined using propidium iodide (uptake) or annexin V-PE (staining).

Previous experiments have shown that an exemplary polynucleotide delivery-enhancing polypeptide, PN73, is an ideal candidate for the treatment of rheumatoid arthritis. FIG. 3 illustrates the ability of several different polynucleotide delivery-enhancing polypeptides to enhance siRNA uptake in cultured monocytes. Transfection using liposomes was used as a comparison. The effect of each peptide on cell viability was also evaluated (figure 4). These data demonstrate the surprising and unexpected discovery that PN73 peptide can transfect human monocytes with high efficiency and low toxicity, suggesting that it is an ideal candidate for the treatment of rheumatoid arthritis in vivo.

Example 5

Enhancing siRNA/delivery by conjugation of siRNA to a polynucleotide delivery-enhancing polypeptide

This example provides the results of a screen to evaluate the activity of siRNA/polynucleotide delivery-enhancing polypeptide conjugates to induce or enhance siRNA uptake in 9L/LacZ cultured cell lines and primary fibroblasts from mice. The materials and methods used in these studies are generally the same as described above, except that siRNA/peptide mixing to produce siRNA/peptide complexes is not required. Table 7 summarizes the percentage uptake of the transfections in 9L/LacZ cells. Table 7 includes the concentrations of the peptides and peptide/siRNA conjugates used. FAM- β -gal labels conjugated to siRNA molecules were used. Table 8 summarizes the results of transfection with MTF. Table 8 includes the peptide/siRNA conjugates used and the concentration of Cy5 label conjugated to the eGFP siRNA molecule.

Table 7:

in LacZ cells, the efficiency of polypeptide-mediated siRNA uptake is enhanced by rationally designed polynucleotide delivery coupled to siRNAs.

Conjugate name	Peptide ID #	peptide/siRNA conjugate concentration	Uptake%
				CoP267nfR137-1	PN267	2.0. mu.M was detected	0％
CoP286nfR138-1	PN286	0.8μM	0％
				CoP287nfR138-1	PN287	0.8μM	0％
CoP284nfR164-1	PN284	1.0. mu.M was detected	0％
				CoP282nfR165-1	PN282	1.0. mu.M was detected	0％
CoP290nfR165-1	PN290	1.0. mu.M was detected	0％
				CoP277nfR167-1	PN73	1.0μM	42.9％
CoP277nfR167-2	PN73	2.0μM	55.4％

Table 8:

polypeptide-mediated efficiency of siRNA uptake in rat tail fibroblasts by rationally designed polynucleotide delivery coupled to siRNAs

Conjugate name	Amino acid sequence	peptide/siRNA conjugate concentration	% intake
				Cy5-dsCoP278nfR270	Maleimide-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-amide (SEQ ID NO: 102)	0.5μM	96.3％
dsCoP277nfR317	Maleimide-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 103)	4μM	83.5％
				dsCoP275nfR321	Maleic acidimide-AAVALLPAVLLALLAPRKKRRQRRRPPQ-amide (SEQ ID NO: 37)	4μM	52.1％
dsCoP285nfR322-1	Maleimide-Dmt-r-FKQqqqqqqqqq-amide (SEQ ID NO: 74)	4uM	41.3％
				dsCoP236nfR332	Maleimide-RQIKIWFQNRRMKWKK-amide (SEQ ID NO: 52)	4μM	36.3％
dsCoP317nfR320	Maleimide-KETWEWTWEWWEWSQPKKKRKV-amide (SEQ ID NO: 104)	2μM	29.6％
				dsCoP316nfR347	Maleimide-RVIRWFQNKRSKDKK-amide (SEQ ID NO: 92)	2μM	17.1％
dsCoP289nfR268	Maleimide-WRFKQqqqqqqqqqqqq-amide (SEQ ID NO: 76)	4μM	3.2％
				dsCoP276nfR319	Maleimide-RKKRRQRRRPPQCAAVALLPAVLLALLAP-amide (SEQ ID NO: 105)	2μM	3.6％
dsCoP298cfR248	NH 2-WRFKC-amide (SEQ ID NO: 106)	4μM	4.1％
				dsCoP280nfR362-1	Maleimide-GRKKRRQRRPPQ-amide (SEQ ID NO: 43)	4μM	1.8％
dsCoP458nfR363-1	Maleimide-KLALKLALKALKAALKLA-amide (SEQ ID NO: 107)	4μM	10.8％
				dsCoP459nfR364-1	Maleimide-GWTLNSAGYLLGKINLKALAALAKKIL-amide (SEQ ID NO: 108)	4μM	54.5％

The previous data indicate that the diversely assembled siRNA/peptide conjugates of the invention efficiently mediate the delivery of siRNAs into different cell types.

Example 6

The delivery of the polynucleotide delivery-enhancing polypeptide in combination with siRNA enhances Knock-down of siRNA gene expression

This example demonstrates that the siRNA/polynucleotide delivery-enhancing polypeptide complexes of the invention effectively knock down the expression of a target gene. In this study, the ability of peptide/siRNA complexes to modulate the expression of human tumor necrosis factor-alpha (hTNF- α) gene, which when overexpressed in human and other mammalian subjects, is examined, suggesting that RA is mediated.

Healthy human blood was purchased from Golden West Biologicals (CA) and Peripheral Blood Mononuclear Cells (PBMCs) were purified from the blood using Ficoll-Pague plus (Amersham) gradient centrifugation. Human monocytes were then purified from PBMCs using microbeads from Miltenyi Biotech. Isolated human monocytes were resuspended in IMDM supplemented with 4mM glutamate, 10% FBS, 1 × nonessential amino acids, and 1 × azure-streptavidin and stored at 4 ℃ until use.

Human monocytes were seeded in 96-well flat bottom plates in 100 μ l of OptiMEM medium (Invitrogen) containing 100,000 cells per well. Transfection reagents were mixed with siRNA at the desired concentrations in OptiMEM medium for 20 minutes (for liposome 2000; Invitrogen) or 5 minutes (for peptides) at room temperature. At the end of the incubation, FBS (final concentration 3%) was added to the mixture and 50. mu.l of this mixture was added to the cells. Cells were incubated at 37 ℃ for 3 hours. After transfection, cells were transferred into a V-plate and pelleted by centrifugation at 1500rpm for 5 minutes. The cells were resuspended in growth medium (IMDM with glutamate, non-essential amino acids and penicillin-streptomycin). After overnight incubation, cells were stimulated with 1ng/ml LPS (Sigma) for 3 hours. After induction, cells were harvested as described above for mRNA quantification and the supernatant was retained for protein quantification.

mRNA was determined using the branched DNA technique (CA) of Genospectra (CA) according to the manufacturer's instructions. To quantify the mRNA levels in cells, mRNA for the housekeeping gene (cypB) and the target gene (TNF-. alpha.) were determined and TNF-. alpha.readings were corrected for cypB to obtain relative luminescence units. To quantify protein levels, TNF-a ELISA from BD Bioscience was used according to the manufacturer's instructions.

As shown in Table 9 below, these siRNAs were directed against different target regions of TNF-. alpha.mRNA. Each oligonucleotide sequence listed in table 9 does not show a 3' overhang (e.g., dNdN, where N represents any nucleotide).

Table 9:

naming and positioning of target sequence of TNF-alpha gene and oligo sequence of siRNA targeting TNF-alpha

ID#	Name of siRNA	Target sequence localization	Oligo sequences	SEQ ID NO：
					N125	TNF-α-1	516-534	GCGUGGAGCUGAGAGAUAA	109
N115	TNF-α-2	430-448	GCCUGUAGCCCAUGUUGUA	110
					N132	TNF-α-3	738-756	GGUAUGAGCCCAUCUAUCU	111
N108	TNF-α-4	360-378	CCAGGGACCUCUCUCUAAU	112
					N138	TNF-α-5	811-829	GCCCGACUAUCUCGACUUU	113
N113	TNF-α-6	424-442	UGACAAGCCUGUAGCCCAU	114
					N143	TNF-α-7	844-862	GGUCUACUUUGGGAUCAUU	115
N107	TNF-α-8	359-377	CCCAGGGACCUCUCUCUAA	116
					N137	LC1	806-828	AAUCGGCCCGACUAUCUCGACUU	117
N122	LC2	514-532	AAUGGCGUGGAGCUGAGAGAU	118
					N130	LC3	673-691	AACCUCCUCUCUGCCAUCAAG	119
N101	LC4	177-195	AACUGAAAGCAUGAUCCGGGA	120
					N140	LC5	820-838	AAUCUCGACUUUGCCGAGUCU	121
N135	LC6	781-799	AAGGGUGACCGACUCAGCGCU	122
					N128	LC7	636-654	AAUCAGCCGCAUCGCCGUCUC	123
N127	LC8	612-630	AACCCAUGUGCUCCUCACCCA	124
					N118	LC9	472-490	AAGCUCCAGUGGCUGAACCGC	125
N111	LC10	398-416	AAGUCAGAUCAUCUUCUCGAA	126
					N110	LC11	363-381	AAGGGACCUCUCUCUAAUCAG	127
N105	LC12	265-287	CCUCAGCCUCUUCUCCUUCCUGA	128
					N104	LC13	264-282	AAUCCUCAGCCUCUUCUCCUU	129
N120	LC14	495-513	AACCAAUGCCCUCCUGGCCAA	130
					N153	LC16	1535-1555	CUGAUUAAGUUGUCUAAACAA	131
N136	LC17	787-807	CCGACUCAGCGCUGAGAUCAA	132
					N152	LC18	1327-1347	CUUGUGAUUAUUUAUUAUUUA	133
N114	LC19	428-448	AAGCCUGUAGCCCAUGUUGUA	134
					N145	LC20	982-1002	UAGGGUCGGAACCCAAGCUUA	135
N101	YC-1	177-195	CUGAAAGCAUGAUCCGGGA	136
					N103	YC-2	251-269	AGGCGGUGCUUGUUCCUCA	137
N106	YC-3	300-318	CCACCACGCUCUUCUGCCU	138
					N109	YC-4	362-380	AGGGACCUCUCUCUAAUCA	139
N113	YC-5	424-442	UGACAAGCCUGUAGCCCAU	140
					N115	YC-6	430-448	GCCUGUAGCCCAUGUUGUA	141
N117	YC-7	435-453	UAGCCCAUGUUGUAGCAAA	142
					N120	YC-8	495-513	CCAAUGCCCUCCUGGCCAA	143
N121	YC-9	510-528	CCAAUGGCGUGGAGCUGAG	144
					N123	YC-10	515-533	GGCGUGGAGCUGAGAGAUA	145
N125	YC-11	516-534	GCGUGGAGCUGAGAGAUAA	146
					N126	YC-12	558-576	GCCUGUACCUCAUCUACUC	147
N130	YC-13	673-691	CCUCCUCUCUGCCAUCAAG	148
					N132	YC-14	738-756	GGUAUGAGCCCAUCUAUCU	149
N133	YC-15	772-790	GCUGGAGAAGGGUGACCGA	150
					N134	YC-16	776-794	GAGAAGGGUGACCGACUCA	151
N136	YC-17	787-807	GCCCGACUAUCUCGACUUU	152
					N141	YC-18	841-859	GCAGGUCUACUUUGGGAUC	153
N143	YC-19	844-862	GGUCUACUUUGGGAUCAUU	154
					N144	YC-20	853-871	UGGGAUCAUUGCCCUGUGA	155

ID#	Name of siRNA	Target sequence localization	Oligo sequences	SEQ ID NO：
					N146	YC-21	985-1003	GGUCGGAACCCAAGCUUAG	156
N147	YC-22	1179-1197	CCAGAAUGCUGCAGGACUU	157
					N148	YC-23	1198-1216	GAGAAGACCUCACCUAGAA	158
N149	YC-24	1200-1218	GAAGACCUCACCUAGAAAU	159
					N150	YC-25	1250-1268	CCAGAUGUUUCCAGACUUC	160
N151	YC-26	1312-1330	CUAUUUAUGUUUGCACUUG	161
					N154	YC-27	1547-1565	UCUAAACAAUGCUGAUUUG	162
N155	YC-28	1568-1585	GACCAACUGUCACUCAUU	163

Tables 10, 11 and 12 illustrate the efficacy of specific oligonucleotide sequences complexed to the polynucleotide delivery-enhancing polypeptides of the present invention to target and knock down the expression level of the TNF- α gene in human monocytes.

Table 10:

percentage of TNF-alpha knockdown mediated by PN73/siRNA complexes

Table 11:

percentage of TNF-alpha knockdown mediated by PN509/siRNA complexes

Table 12:

percentage of TNF-alpha knockdown mediated by PN250/siRNA complexes

The foregoing results (tables 10, 11 and 12) demonstrate that effective levels of knockdown of TNF- α gene expression can be achieved in mammalian cells using the novel siRNA/polynucleotide delivery-enhancing polypeptide compositions of the present invention.

Screening and characterization

Human monocytes treated with LPS (CD14+) induced TNF-. alpha. -specific mRNA within about 2 hours, and the protein level of TNF-. alpha.peaked in the next 2 hours. siRNA candidate sequences were transfected into monocytes using liposome 2000 and then the infected cells were treated with LPS and TNF- α mRNA levels were measured after approximately 16 hours and siRNA were screened for knockdown activity. 56 siRNA sequences were designed and screened for their level of knockdown of TNF- α mRNA and protein in activated human primary monocytes. A representative group of 27 siRNA sequences had activities ranging from 80% mRNA knock-down activity to no detectable activity. Typically, TNF- α protein levels are reduced more than mRNA levels, e.g., a 50% knock-down of TNF- α mRNA (TNF- α -1) results in a 75% reduction in TNF- α protein levels. Selected siRNAs were obtained showing dose response at knockdown levels from 30% to 60%Curve line. Calculated IC₅₀Values were from 10pM to 200 pM. Although the siRNA sequences evaluated were distributed throughout the TNF- α transcript, the most potent siRNAs identified were located in two regions: the middle of the coding region and the 3' -UTR.

Example 7

Delivery enhancing polypeptide of polynucleotide conjugated to siRNA enhances siRNA Knock-down of gene expression

This example demonstrates that the peptide-siRNA conjugates of the invention knock down the expression of the target gene. The materials and methods in these studies were the same as those described above, except that no mixing of siRNA and peptide was required. In this series of studies, the knockdown experiments included a comparison of peptide/siRNA-mediated knockdown in the presence and absence of liposomes. Table 13 below illustrates the results of this example.

Table 13:

peptide/siRNA-mediated knockdown of TNF-alpha Gene expression in the Presence and absence of liposomes

The foregoing data (Table 13) indicate that a diversely assembled polynucleotide delivery-enhancing polypeptide of the present invention coupled to siRNAs can enhance siRNA-mediated knockdown of TNF- α gene expression in a mammalian subject.

Example 8

Time course of siRNA gene expression knockdown

This example presents studies related to the time course of siRNA-mediated knockdown of gene expression. To examine the duration of the siRNA effect, the siRNA transfection protocol mentioned above was used except that fibroblasts from eGFP-expressing mice were used. The transfection reagent used here is a liposome. Cells were replated at day 18 due to overgrowth. The second transfection was performed on day 19 after the first transfection. On day 19 post-transfection, the level of eGFP was determined. GFPI siRNA (GFPI) and hairpin siRNA (D #21) were used together with control pseudo or nonsense siRNA (Qiagen). Knockdown activity was corrected with mock siRNA (Qiagen control). Higher values indicate higher knockdown activity.

Table 14:

time course for knocking down TNF-alpha gene expression by siRNA transfection mediated by liposome

Previous studies (table 14) showed that siRNA knockdown activity became apparent around day 3 and continued to day 9, while target gene expression returned to baseline levels around day 17. Another reduction in eGFP expression occurred after the second transfection at day 18, suggesting that the agent may be repeatedly administered to the cells to produce repeated or persistent knockdown of gene expression.

Example 9

SiRNA-mediated knockdown of TNF-alpha gene expression complexed with a polynucleotide delivery-enhancing polypeptide Dose dependence

This example demonstrates that siRNA-mediated knockdown activity complexed with the exemplary polynucleotide delivery-enhancing polypeptide PN73 in activated human monocytes is dependent on the dose concentration of the peptide-siRNA complex.

The PN73/siRNA complex ratio provided was constant, with a ratio between PN73 and siRNA of about PN 73: siRNA to 82: 1 (table 15). 400 Nameolar siRNA was complexed with 33. mu.M PN73 in OptiMEM medium for 5 minutes. After compounding, the complex was serially diluted with OptiMEM (1: 2 ratio). The complex is added to human monocytes for transfection. Subsequent induction and quantitation of mRNA was done as described above.

Table 15:

dose-dependent peptides for TNF-alpha Gene expression knockdown

In a related series of experiments, siRNA was serially diluted and combined with a fixed amount of PN73(1.67 μ M). Alternatively, the PN73 polynucleotide delivery-enhancing polypeptide is complexed with a titrated amount of siRNA. PN73 (1.67. mu.M) was complexed with each titration of LC20siRNA in OptiMEM medium for 5 minutes at room temperature. After complexing, the complex is added to human monocytes for transfection. The data for induction and mRNA quantification provided in table 16 below were obtained by the methods described hereinabove.

Table 16:

siRNA dose dependent TNF-alpha gene expression knockdown

siRNA concentration (nM)	Control (%)	TNF-alpha Gene expression (%)
			0.8nM	100.0％	84.7％
4nM	100.0％	59.4％
			20nM	100.0％	65.2％
100nM	100.0％	54.7％

Example 10

Multiple dosing regimen to extend siRNA knockdown effects in mammalian cells

This example demonstrates that a multiple dosing regimen will effectively amplify the knockdown effect of mammalian cell gene expression mediated by the siRNA/polynucleotide delivery-enhancing polypeptides of the invention. The materials and methods used for these studies were the same as described above, except that repeated transfections were performed at the indicated times. Sham siRNA (Qiagen) was used as a side-by-side control. Table 17 summarizes the data for multiple transfections with peptide/siRNA complexes. Percent TNF- α gene knockdown activity represents the percent of total gene expression.

Table 17:

TNF-alpha gene expression knockdown Activity following multiple transfection with peptide/siRNA complexes

The foregoing results (Table 17) indicate that the TNF-. alpha.gene expression knockdown effect in mammalian cells can be maintained or re-induced when multiple transfections are performed at the appropriate time (in this case, between about day 5 and 7 after the first transfection).

Example 11

In vivo siRNA/peptide-mediated TNF-alpha gene expression knockdown activity

This example provides in vivo data demonstrating the efficacy of siRNA/polynucleotide delivery-enhancing polypeptide compositions of the invention to mediate systemic delivery and therapeutic gene knockdown by siRNA, which compositions effectively modulate target gene expression and therapeutically alter the phenotype of cells.

5-week-old mice expressing human TNF-. alpha.were purchased from Hellenic Pasteur institute, Greece). Mice were given intravenously 300 μ l of physiological saline twice a week (4 mice) with RA drug like gram (Ramicade) (5mg/kg) once a week (2 mice), or with LC20siRNA mixed with PN73 at a 1: 5 molar ratio (2mg/kg) twice a week (2 mice). During the injection period, plasma samples were collected for ELISA testing (R & D Systems) while paw scores were performed twice a week as a recognized indicator of RA disease progression and treatment efficacy. The plasma levels of TNF- α protein from the treated mice are shown in Table 18 below.

Table 18:

amount of TNF-alpha protein in plasma measured by ELISA

^*These data represent the mean values in pg/ml for the experimental mice.

The above results indicate that in peptide/siRNA-treated mice, circulating TNF- α protein levels are effectively reduced compared to levels in mock or saline (control) treated mice.

Additional evidence for in vivo efficacy of the siRNA/polynucleotide delivery-enhancing polypeptide compositions and methods of the invention was obtained from the murine subjects undergoing paw scoring as described above (a recognized indicator of RA disease status and efficacy). Due to the different starting points (some animals scored earlier), the scores for all animals in the experiment were adjusted to 0. Each paw was given a score of 0 to 3, with a maximum of 12, according to the scoring index below.

0: is normal

1: edema or deformity of the paw or ankle

2: deformity of the paw and ankle joint

3: stiffness of the wrist or ankle joint

The results of these paw score evaluations are presented graphically in fig. 5. These data indicate that when the polynucleotide delivery-enhancing polypeptide PN73 is injected into animals, therapeutic amounts of siRNAs (e.g., LC20, TNF- α 2, and TNF- α 9(UAGCCCAUGUUGUAGCAAA (SEQ id No.187))) can be delivered, as shown by the delayed progression of RA at week 8. At week 8, PN73/siRNA treated mice gave better results in paw score than the gram-like treated mice. When paw score assessments were performed 11 weeks after treatment, PN73/LC20 complex gave paw score assessments comparable to those of the akoid-treated mice. At a 1: 5 ratio of PN73 peptide/LC 20siRNA, 2mg/kg LC20 achieved the greatest relative observed delay in RA progression compared to the lower dose of LC20 tested. Table 19 below summarizes the relative effects of several siRNAs in 5 different groups evaluated after treatment with PN73 and siRNAs.

Table 19:

group summary

Group mark	Treatment of＊	Relative Effect of SiRNA
			TNF#1	LC20, class g, PBS	LC20 has the same effect as Ramicade
TNF#4	LC20 and LC13	Total low paw score
			TNF#5	LC20 coupled to PN73	Overall low paw score; the conjugate has lower activity than the complex
TNF#6	LC20, YC12 and LC17	Overall low paw scores. YC12 and YC17 are not as efficient as LC20
			TNF #7 (FIG. 5)	LC20, TNF-alpha 2 and TNF-alpha 9	By week 8, LC20 and TNF- α 9 were more potent than nanograms; by week 11, LC20 performed as a gram-like effect

^*Testing siRNAs in the presence or absence of PN 73; class gram (Ramicade) is a positive treatment control; PBS is a negative treatment control.

The above results indicate that the siRNA and polynucleotide delivery-enhancing polypeptide compositions of the present invention provide promising new therapeutic tools for modulating gene expression and treating and managing diseases. siRNAs of the invention, e.g., siRNAs that target human TNF- α -specific mRNAs to degrade, provide higher specificity, lower immunogenicity, and better disease amelioration than existing small molecule, soluble receptor or antibody treatments for RA. More than 50 candidate siRNA sequences were selected that target hTNF- α and produced 30 to 85% knockdown with a single dose. Comparing the mononuclear cellular fluorescent RNA uptake of more than 20 computer-designed peptide complexes and/or covalent molecules, many were found to have significantly stronger uptake, IC's, than liposomes or cholesterol-conjugated siRNA₅₀The value was < 10 pM. In vitro activated human monocytes, the peptide-siRNA formulations effectively knock down TNF- α mRNA and protein levels.

An exemplary candidate delivery peptide/siRNA formulation was evaluated in two transgenic mouse models constitutively expressing human TNF- α. Animals treated with IV injection of 2mg/kg siRNA or with infliximab twice a week at the beginning of 6 weeks of age showed stability of RA scores (paw and joint inflammation) at the beginning of 7 weeks of age, compared to controls where the disease state persisted up to 11 weeks. At 9 weeks of age, the reduction in RA scores for siRNA treated animals was similar to that of the infliximab treated animals, but the plasma TNF-alpha protein levels were significantly lower than those of the infliximab treated animals.

Based on the present disclosure, the use of sirnas that inhibit the expression of target genes (e.g., cytokines such as TNF-a) that play an important role in pathological conditions such as inflammation provides an effective treatment to alleviate or prevent disease symptoms such as RA in a mammalian subject. Exemplary peptide/siRNA compositions used within the scope of the methods and compositions of the present invention provide advantages associated with their ability to reduce or eliminate target gene expression (e.g., TNF- α expression) rather than complexing with a target gene product (e.g., TNF- α) as with antibodies or soluble receptors.

According to the teachings of the present invention, improved systemic administration of nucleic acids provides another advantage in the development of siRNAs as drugs. Specific challenges in this context include delivering siRNA to the target cell or tissue through a tissue barrier, maintaining the stability of the siRNA and intracellular delivery of siRNAs across the cell membrane into the cell in sufficient effective amounts. The present disclosure demonstrates for the first time an effective in vivo delivery system comprising a novel peptide/siRNA composition targeting specific gene expression, such as human TNF-alpha expression, that attenuates disease activity in transgenic animal models predictive of the target disease, as exemplified by the study of the RA murine model. In a related study, the compositions and methods of the invention effectively inhibited the expression of TNF- α in activated monocytes from RA patients. These studies suggest that the RNAi pathway effectively mediates alterations in cellular phenotype and disease progression through intracellular effects on the TNF-pathway, and avoids toxic effects due to antibody/TNF-alpha complexes with residual immunoreactivity in the circulation, which characterize current RA antibody therapies. In particular, all tests herein had the lowest associated toxic effects, such that the doses of siNAs and polynucleotide delivery-enhancing polypeptides shown in these examples were collectively associated with cell viability levels of at least 80-90% or greater.

Example 12

Rational design for optimized polynucleotide delivery-enhancing polypeptides

This example provides exemplary designs and data for optimizing a polynucleotide delivery-enhancing polypeptide of the invention. The rational design procedure was performed with histone H2B-derived polynucleotide delivery-enhancing polypeptide.

Table 20:

deletion and modification of PN73

Table 20 provides a graph of the original structure of PN73 and the PN73 derivatives generated for the rational design of optimized PN 73-based polynucleotide delivery enhancing polypeptides. The grey-painted C-terminus of each peptide represents the hydrophobic region of the peptide, and the black-painted N-terminus of each peptide represents the hydrophilic region. The parent peptide PN73 shown above is an example of a polynucleotide delivery-enhancing polypeptide that induces or enhances siRNA delivery into a cell.

To better understand the function-structure activity relationship of this and other polynucleotide delivery-enhancing polypeptides, primary structure studies were performed by characterizing the C-and N-terminal functions as well as the activity of PN73 and other chemical moiety conjugates.

The amino acid sequence of human histone 2B (H2B) is shown below.

PN73, PN360, and PN361 are peptide fragments of H2B, and the portions of the H2B protein they represent are indicated in parentheses following the peptide names below. The amino acid sequences of PN360 and PN361 listed below are aligned with the corresponding sequences found within PN 73. The PN73 peptide fragment in the H2B amino acid sequence is underlined, representing amino acids 13 to 48 of H2B. It may also be amino acids 12 to 48 of H2B. PN360 shares the N-terminus with PN73 but lacks the C-terminus of PN73, while PN361 shares the C-terminus with PN73 but lacks the N-terminus of PN 73. The PN73 conjugate is PN73 covalently linked to a single siRNA strand (e.g., the sense strand). PN404 is a version of PN73 in which all lysines have been replaced by arginines, and PN509 is a pegylated PN73(PEG molecular weight is 1 kdalton) derivative, the N-terminus of which is pegylated.

H2B (Histone 2B) amino acid sequence

MPEPAKSAPAPKKGSKKAVTKAQKKDSKKPKRSRKESYSVYVY

KVLKVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRST

ITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSSK(SEQ ID NO：164)

PN73(13-48)

NH 2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 59)

PN360 (13-35; N-terminal of PN73)

NH 2-KGSKKAVTKAQKKDGKKRKRSRK-amide (SEQ ID NO: 57)

PN361 (24-48; C-terminal of PN73)

NH 2-KKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 58)

PN73(13-48) -siRNA (sense strand) conjugates

siRNA-KGSKKAVTKAQKKDGKKJ1 KRSRKESYSVYVYKVLKQ-amide (SEQ ID NO 59)

PN404 (PN 73 with all lysines replaced by arginines)

NH 2-RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ-amide (SEQ ID NO: 91)

PN509 (Pegylated PN73)

PEG-RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ-amide (SEQ ID NO: 90).

Figure 6 provides the results of the aforementioned rationally designed PN 73-derived polynucleotide delivery-enhancing polypeptide uptake efficacy and cell viability studies in mouse tail fibroblasts. The activity change of modified PN73 in mouse tail fibroblasts is illustrated by the legend. Unlike PN404, PN509 increases uptake without increasing toxicity. Deletion of the N-terminal residue of PN73 reduces activity, while removal of the C-terminal residue abolishes activity. Both PN73 and PN509 showed higher activity in primary cells than liposomes (Invitrogen, CA).

Example 13

AcetylatedStability enhancement of polypeptides in plasma by polynucleotide delivery

The purpose of this example was to determine whether modification of the exemplary polynucleotide delivery-enhancing polypeptide PN73 would increase the stability of the peptide and thereby its transfection activity. The stability of the unmodified, N-terminally pegylated and N-terminally acetylated forms of PN73 in plasma was compared. The C-terminus of PN73 is amidated. Exclusion chromatography in combination with a uv detector was used to study the characteristics of the stability of the unmodified and modified forms of PN73 before and after plasma incubation.

In the absence of plasma, the unmodified, pegylated and acetylated forms of PN73 showed distinct but overlapping UV traces at approximately 9 minutes. However, after 4 hours of plasma exposure, UV traces specific to unmodified PN73 and pegylated PN73 were no longer present, suggesting that significant degradation occurred. In contrast, the unique UV trace of acetylated PN73 remained, suggesting that this modification significantly increased the stability of PN73 in the plasmid compared to the unmodified and pegylated PN73 form.

These data show the surprising and unexpected discovery that the stability of PN73 in plasma can be enhanced by N-terminal acetylation of PN73 peptide. The primary structure of the acetylated PN73 peptide is as follows:

Ac-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 59)

Example 14

The polynucleotide delivery-enhancing polypeptide does not elicit an interferon response

The purpose of this example is to compare the interferon response of cells transfected with siRNA with the polypeptide PN73 peptide enhanced by liposomal reagents and siRNA or exemplary polynucleotide delivery. The interferon response was detected by ELISA (protein) and bDNA (mRNA levels).

Typically, siRNA molecules are delivered into cells by liposome-mediated transfection. However, this often results in poor delivery efficiency, an in vivo inflammatory response, and upregulation of interferon gene expression that inhibits cell growth. Therefore, liposome-mediated transfection has limited reduction of target gene expression levels, making siRNA an ineffective therapeutic approach and a tool for gene expression studies. Delivery of siRNA by PN73 overcomes this problem.

FIG. 7 provides the results of liposome and PN73 peptide-transfected bDNA analysis of several different siRNAs. siRNAs at concentrations of 1nM, 10nM, 100nM or 200nM bind to liposomes or PN 73. Interleukin 1 β (IL-1 β) was used as a molecular marker to determine the interferon response, and Qneg was used as a negative control. As shown in FIG. 7, liposomes complexed with 100nM or 200nM TNF- α 9 siRNA resulted in a significant increase in IL-1 β mRNA levels. Moreover, all other siRNAs tested resulted in a slight increase in IL-1. beta. mRNA levels. In contrast, the same siRNAs complexed with PN73 peptide did not result in increased IL-I β mRNA levels.

To further characterize the differences in interferon responses observed between cells transfected with liposomes and cells transfected with PN73, the protein expression levels of the following molecular markers were determined using an ELISA assay: interleukin 1 β (IL-1 β), interferon- α (INF- α), interleukin 6(IL-6), interleukin 8(IL-8), interleukin 12(IL-12), MIP-1 α, interferon- γ (IFN- γ), and tumor necrosis factor- α (TNF- α). Table 21 summarizes the relative protein expression levels of Lipofectamine conjugated to siRNA or PN73 transfected cells conjugated to siRNA.

Table 21:

relative protein expression levels of interferon-responsive molecular markers

As presented in table 21, no matter what transfection reagent was used, neither siRNA LC20 nor LC17 responded to interferon. However, transfection of IFN-1 or TNF- α 9 with liposomes resulted in increased expression levels of IL-1 β, IL-6, and MIP-1 α proteins. In contrast, transfection of all the siRNAs tested with PN73 did not induce observable protein expression of any of the interferon response markers tested.

These data from ELISA assays show the surprising and unexpected finding that PN 73-mediated transfection of siRNAs does not elicit an interferon response.

Example 15

siRNA conjugated to a polynucleotide delivery-enhancing polypeptide in comparison to a polynucleotide delivery-enhancing polypeptide Complexed siRNA provides enhanced knockdown activity

The purpose of this example was to compare the knockdown activity of the polypeptide PN73 or siRNAs LC13 and LC20 coupled or bound to exemplary polynucleotide delivery-enhancing polypeptides in human monocytes. The isolation and transfection of human monocytes and methods for measuring knockdown activity have been discussed previously. Qneg represents random siRNA sequences, which served as negative controls in these experiments. The observed Qneg knockdown activity was corrected to 100% (100% gene expression level) and the activity of LC20 and LC13 was expressed as a relative percentage of negative controls. LC20 and LC13 are siRNAs targeting the human TNF- α gene. FIG. 8 shows the knockdown activity of siRNAs LC20 and LC13 in the absence of PN73 (FIG. 8-C), complexed to PN73 (FIG. 8-B), or conjugated to PN73 (FIG. 8-A). The concentrations of LC20 and LC13 tested ranged from 0nM to 2.5 nM. PN73 was maintained in a 1: 1 ratio in both complex and conjugate experiments.

As expected, LC20 and LC13 showed little knock-down activity in the absence of PN73 (fig. 8-C). LC13 and LC20 resulted in approximately 15% and 30% reduction in TNF-a gene expression relative to Qneg control when complexed with PN73 (fig. 8-B). However, when siRNA was conjugated to PN73, the TNF- α knockdown activity decreased below 60% (fig. 8-a). This is a significant increase in knockdown activity of the siRNA compared to the PN73/siRNA complex. Thus, these data show the surprising and unexpected discovery that siRNA knockdown activity is significantly enhanced when siRNA is conjugated to the exemplary polynucleotide delivery-enhancing polypeptide PN 13.

Example 16

The polynucleotide delivery-enhancing polypeptide rescues serum-inhibited cholesterol Enhanced siRNA uptake

This example demonstrates that the addition of a membrane-penetrating peptide to a delivery formulation comprising an siRNA conjugated to a cholesterol moiety reduces the inhibitory effect of serum on cholesterol-siRNA uptake in a dose-dependent manner. To analyze siRNA uptake, cells were washed with PBS, trypsinized (only to adherent cells), and then analyzed by flow cytometry. Uptake of the siRNA designated BA described above was also measured by intracellular Cy5 or FAM fluorescence intensity, and cell viability was assessed by addition of propidium iodide or annexin v-PE. To distinguish cellular uptake from membrane insertion of fluorescently labeled siRNA, the fluorescence of the cell membrane surface was quenched with trypan blue.

Table 22:

PN 73-mediated transfection rescued serum-induced inhibition of cellular uptake of cholesterol-conjugated siRNA as measured by Mean Fluorescence Intensity (MFI)

Percentage of serum	Cholesterol alone siRNA (MFI)	Unconjugated siRNA (MFI) with 20. mu. MPN73
			0	24.8	32.9
5％	1.55	11.5
			10％	1.34	6.39
20％	1.19	5.85

The data in Table 22 show that the presence of serum significantly reduces cellular uptake of siRNA conjugated to the cholesterol moiety of the invention. However, in the presence of the exemplified delivery enhancing peptide PN73, the cellular uptake of unconjugated siRNA was rescued.

Cellular uptake of cholesterol-conjugated siRNA of the invention (cholesterol siRNA + PN73) complexed with the transmembrane peptide delivery enhancer PN73, and unconjugated siRNA (siRNA + PN73) complexed with PN73 were compared. As shown in FIG. 9, unconjugated siRNA or cholesterol conjugated siRNA gave the same cellular uptake activity at high concentrations of PN 73. For these and related uptake assays, cholesterol-coupled siRNA and siRNA/PN73 complexes were transfected into the same cells as described above in Opti-Mouse Tail Fibroblasts (MTF) in culture medium were added with fixed or varying concentrations of serum. The final concentration of siRNA for cholesterol and complexes was 0.2. mu.M. Uptake efficiency and mean fluorescence intensity were assessed by flow cytometry.

The ability of PN73 and additional PN250 to rescue inhibition of cellular uptake of cholesterol-conjugated siRNA was further characterized in fibroblasts (MTF) cells at the tail of the mouse. FIG. 10 illustrates the effect of increasing concentrations of Fetal Bovine Serum (FBS) on siRNA cell uptake. Without PN73 or PN250, cellular uptake of cholesterol-conjugated siRNA was significantly reduced in the presence of only 5% FBS. However, in the presence of 40 μ M PN73 or 80 μ M PN250, the uptake of siRNA was rescued.

The previous data (table 22 and fig. 10) show that siRNAs conjugated to cholesterol significantly enhanced their cellular uptake. However, the presence of serum greatly reduces or even eliminates the uptake of cholesterol-coupled siRNAs. This is probably due to the binding of cholesterol moieties to serum proteins that inhibit the ability of cholesterol-binding siRNAs to enter target cells. However, in the presence of delivery enhancing agents, such as the membrane penetrating peptides PN73 and PN250, the serum-inhibited cellular uptake is rescued. More specifically, the addition of a membrane-penetrating peptide to a delivery formulation comprising an siRNA conjugated to a cholesterol moiety reduces the inhibitory effect of serum on the dose-dependent manner of cholesterol-siRNA uptake.

Example 17

Deletion analysis of exemplary polynucleotide delivery-enhancing Polypeptides

This example illustrates the design of experiments that optimize siRNA cellular uptake and siRNA-mediated target gene knockdown activity of the exemplary polynucleotide delivery-enhancing polypeptide PN 73.

Table 23 below shows exemplary polynucleotide delivery-enhancing polypeptide PN73 and truncated derivatives thereof. The amino acid sequences of PN360 and PN361 listed below are identical to the corresponding amino acid sequence of PN 73. PN360 shares the N-terminus with PN73 but lacks the C-terminus of PN73, while PN361 shares the C-terminus with PN73 but lacks the N-terminus of PN 73. PN766 corresponds to the 15 amino acids at the end of PN 73C. PN73, PN360, PN361 and PN766 do not carry a C-terminal FITC (fluorescein isothiocyanate) (i.e., -GK [ EPSILON ] G-amide) label. Table 23 further shows 11 truncated forms of the exemplary polynucleotide delivery-enhancing polypeptide PN73, which were generated by the sequential deletion of 3 residues from the N-terminus of the PN73 peptide, with the exception of PN 768. All of these peptides carry a C-terminal FITC (fluorescein isothiocyanate-5-isothiocyanate) label (i.e., -GK [ EPSILON ] G-amide), so cells containing the peptide can be detected by fluorescence microscopy or sorted by flow cytometry. Notably, PN766 and PN708 have the same amino acid sequence, except PN708 has a C-terminal FITC tag. The original structure and truncated form of PN73 for which transfection activity is to be detected are described below.

Table 23:

PN73 missing series

Example 18

Deletion analysis of exemplary polynucleotide delivery-enhancing Polypeptides

The functional domain of the exemplary polynucleotide delivery-enhancing polypeptide PN73 is critical to the ability of the polynucleotide delivery-enhancing polypeptide to efficiently deliver siNAs into cells. These domains include membrane adhesion, fusion and nucleotide binding regions. Briefly, membrane adhesion describes the ability of exemplary polynucleotide delivery enhancing polypeptides to bind to cell membranes. The fusion properties reflect the ability to dissociate from the cell membrane and enter the cytoplasm. The membrane adhesion and fusion domains of the peptide are mechanistically closely linked (i.e., the ability of the peptide to enter cells) and therefore difficult to separate in an experiment. Thus, these two properties (domains of the peptide) are combined in one assay. The methods employed to define each of the above domains of a polynucleotide delivery-enhancing polypeptide are discussed in more detail below. Finally, nucleic acid binding describes the ability of the peptide to bind nucleotides. Table 24 summarizes data identifying the membrane adhesion/fusion and nucleotide binding domains of the exemplary polynucleotide delivery-enhancing polypeptide PN73 (data for all concentrations tested are not shown).

Table 24:

summary of domain analysis of the PN73 peptide deletion series

NT is not detected; the peptide concentrations given (in parentheses) are those that achieve the given percent uptake or relative value MFI.

Exemplary polynucleotide delivery-enhancing polypeptide membrane adhesion and fusion domains:

the efficacy of exemplary polynucleotide delivery enhancing full-length and truncated forms of polypeptide PN73 into cells was examined ex vivo by cellular uptake assays using primary Mouse Tail Fibroblast (MTF) cells. The number of cultured cells that received the FITC-labeled peptide was measured by flow cytometry. The percentage of peptides taken up by the cells is expressed relative to the total number of cells in culture. In addition, the amount of FITC-labeled peptide found in the cells was evaluated using the Mean Fluorescence Intensity (MFI). MFI is directly related to the amount of intracellular FITC-labeled peptide: higher relative MFI values correspond to higher amounts of intracellular FITC-labeled peptide. Peptides in Table 23 were evaluated at concentrations of 0.63. mu.M, 2.5. mu.M and 10. mu.M; PN768 was detected at 2. mu.M, 10. mu.M and 50. mu.M.

The day prior to transfection, full-length and truncated forms of the exemplary polynucleotide delivery-enhancing polypeptide PN73 were exposed to cells. FITC-tagged peptide at RT in Opti-The cells were diluted in medium (Invitrogen) for about 5 minutes and then added to the cells. Cells were transfected for 3 hours, washed with PBS, trypsinized, and then analyzed by flow cytometry. Cell viability was determined as described above. The fluorescence at the cell membrane surface was quenched with trypan blue to differentiate cellular uptake from membrane inserts.

For cellular uptake analysis, all concentrations tested of full-length FITC-labeled PN73 peptide (PN690) achieved almost 100% cellular uptake (10 μ M results are shown in table 24 under the designation "% peptide cellular uptake"). The remaining truncated form of PN73, which reached a percentage cellular uptake (value in parentheses) at 10 μ M concentration (except PN768, which required 50 μ M) similar to that of PN690, suggesting that the N-terminus of PN73 is not essential for the ability of the peptide to enter cells. An exemplary polynucleotide delivery enhancing polypeptide identified as PN 768C-terminal 5 residues of PN73 are sufficient for cellular uptake of the peptide. However, notably and not shown in table 24, at 0.63 μ M the truncated form of PN73 showed a decrease in cellular uptake activity proportional to the length of the peptide. In other words, the general observation of the tested peptide at a concentration of 0.63 μ M was that its cellular uptake activity decreased as the length of the PN73 peptide decreased, suggesting that the cellular peptide uptake activity was dose dependent.

These results indicate that all truncated forms of the exemplary polynucleotide delivery-enhancing polypeptides retain the ability to enter a high percentage of cultured cells.

Although cellular uptake assays indicated that truncated forms of the exemplary polynucleotide delivery-enhancing polypeptide PN73 had similar cellular uptake activity, MFI measurements based on the average amount of FITC-labeled peptide entering the cells showed that the truncated forms of P73 were distinct. As in the column of table 24 entitled "peptide FITC MIF", the MFI of the full-length FTCC-labeled PN73 peptide (PN690) showed a dose-dependent increase, reaching the highest MFI at 10 μ M, which was approximately 125 units. The MFI levels of PN66, PN685, and PN660 are similar to the MFI of full-length PN73(PN 690). However, the MFI levels for PN735 and PN655 decrease, being 80MFI units and 60MFI units, respectively. At the same time, a significant decrease in MFI detection was observed for PN654, PN708, PN653, PN652, PN651, and PN768, suggesting that the deletion of the 18N-terminal residues of PN73 abolished efficient uptake of this peptide by the cells.

These results indicate that all truncated forms of PN73 can enter a high percentage of cultured cells, and in particular, the first 18 residues at the N-terminus of the exemplary polynucleotide delivery-enhancing polypeptide PN73 are necessary for efficient uptake of the peptide by cultured cells. These results indicate that the exemplary polynucleotide delivery-enhancing polypeptides truncated derivatives of PN73(PN 661; PN 685; PN 660; PN735 and PN655) are able to efficiently enter cultured cells.

Taken together, the data for cellular uptake of the peptide indicates that the C-terminal 5 residues of the exemplary polynucleotide delivery-enhancing polypeptide PN73 are sufficient to enter the cell, suggesting that the membrane adhesion/fusion domain of the peptide is located C-terminally. However, the peptide FITC MFI data indicates that removal of the 18 residues N-terminal of the exemplified polynucleotide delivery-enhancing polypeptide PN73 limits the efficiency of entry of the peptide into cells, suggesting that the N-terminal of the peptide is essential for efficient uptake characteristics of the membrane adhesion/fusion domain.

The nucleotide binding domain of an exemplary polynucleotide delivery-enhancing polypeptide:

the ability of each peptide of the deletion series to complex and deliver siRNA into primary MTF cells was measured by cellular uptake analysis and MFI. The identity of the nucleotide binding domain of the exemplary polynucleotide delivery-enhancing polypeptide PN73 is shown by comparing the relative amounts of siRNA taken up by cells lacking the series of different peptides. The peptides either showed a high percentage of siRNA cellular uptake (40% or higher), which was correlated with the presence of the nucleotide binding domain. A low percentage of siRNA cellular uptake (below 30%) reflects the lack of nucleotide binding domains.

To detect full-length and truncated forms of the exemplary polynucleotide delivery-enhancing polypeptide PN73, MTF cells were treated with siRNA and peptide coupled to Cy5 or FAM as described above. After cell washing, cells were treated with pancreatin and then analyzed by flow cytometry. Intracellular uptake of siRNA was measured using intracellular Cy5 fluorescence intensity; all fluorescence at the cell membrane surface was quenched with trypan blue. Uptake is expressed as a value relative to the total number of cells.

Table 24 shows the results of the percentage cellular uptake of siRNA by peptide coupled to 0.5 μ M Cy 5. non-FITC labeled PN73(PN643) reached near 100% uptake at 10 μ M concentration (data not shown). However, when the PN73 peptide was labeled with the FITC (PN690) tag, its maximum cellular uptake activity observed at 2.5 μ M was reduced to about 60%, suggesting that the addition of the FITC tag interfered with the cellular uptake activity of the peptide. Thus, the cellular uptake activity observed for each peptide in the present assay may not reflect the actual cellular uptake activity of the siRNA by that peptide. However, as indicated by PN661, a slight decrease in cellular uptake activity of siRNA was observed when the extreme 3 residues of the N-terminus of PN73(PN690) were removed. Similarly, both PN660 and PN708 had a moderate decrease in siRNA cellular uptake activity compared to full-length PN73(PN690),

these data indicate that the polynucleotide delivery-enhancing polypeptides of the invention, including PN661, PN660, and PN708, are capable of binding nucleic acids. In contrast, a decrease in siRNA uptake activity was observed for PN685, PN735, PN655 and PN 654. No significant siRNA uptake activity was observed for PN653, PN652 or PN651, indicating that the C-terminal 12 amino acids of the exemplary polynucleotide delivery-enhancing polypeptide (37-48 residues of H2B protein) did not contain a nucleotide binding domain. In addition, since the PN661, PN660, and PN708 peptides do not include the three residue deletion of the full length peptide (PN690), the noted siRNA binding capacity is still retained. These data indicate that the nucleotide binding domain present at the N-terminus of an exemplary polynucleotide delivery-enhancing polypeptide is sensitive to the presence of specific residues at the N-terminus to bind nucleic acids.

Taken together, these data indicate that the cellular uptake activity of siRNA of the PN73 deletion series suggests that PN708 (residues 34-48) is the minimal C-terminal fragment of PN73 necessary for cellular uptake activity of siRNA. This is consistent with the nucleotide binding domain of the exemplary polynucleotide delivery-enhancing polypeptide PN73 being located at the first 24 residues N-terminal.

The ability of PN73 peptide deletion series to transfect siRNAs into cells was further characterized by MFI, which determines the relative average amount of Cy 5-coupled siRNA that entered the cells. Delivery of Cy 5-conjugated siRNA with full-length FITC-labeled PN73 peptide (PN690) achieved an MFI of about 50 relative units. The PN73 deletion series of PN735, PN655, PN654 and PN708 peptides showed reduced MFI, ranging from about 34 units to 44 units. The MFI levels for PN661, PN685 and PN660 of the PN73 deletion series were slightly higher than for the full-length PN73(PN690) peptide. In contrast, PN654, PN653, PN652, and PN651 had relatively little or no MFI (3-14 units), suggesting that little or no siRNA coupled to Cy5 entered the cells after transfection with these peptides. The low MFI values observed at PN653, PN652, and PN651 correlate with data showing that PN654, PN653, PN652, and PN651 do not complex with siRNA or facilitate cellular uptake of siRNA into cells.

Cellular localization of Cy 5-labeled siRNAs complexed with the polynucleotide delivery-enhancing polypeptide PN73 and siRNAs transfected with liposomes (Invitrogen) was compared using fluorescence microscopy imaging. Localization characteristics of siRNA delivered with liposomes were more punctate staining, indicating that there may be localization of endosomes, while PN73 showed more uniform perinuclear staining. The siRNA located in the endosome is not easily accessible to the cytoplasmic RISC complex and is unable to silence the expression of the target gene. In contrast, the uniform cytoplasmic distribution of siRNA observed with PN 73-mediated delivery is a prerequisite to access the RISC complex and to reduce target gene expression. These results indicate that the polynucleotide delivery-enhancing polypeptides of the invention greatly improve siRNA delivery and greatly improve target gene silencing (knockdown) compared to cationic lipids.

These data indicate that the ability of the exemplary polynucleotide delivery-enhancing polypeptide PN73 to enhance the efficient delivery of siRNA into cells depends on the extreme 24 residues of the N-terminus of the peptide. These data indicate that shorter derivatives of the exemplary polynucleotide delivery-enhancing polypeptide PN73(PN 661; PN 685; PN 660; PN 735; PN655 and PN708) are effective for complexing with siRNA and delivering it into cells.

Analysis of truncated forms of polynucleotide delivery-enhancing polypeptides PN360 and PN 361:

the functional-structural activity relationship of the C-terminal and N-terminal regions of PN73 was shown by characterizing PN360 (C-terminal) and PN361 (N-terminal) in the siRNA cellular uptake assay described above.

Table 24 shows that deletion of part of the N-terminus of PN73 (see PN361) reduces siRNA cellular uptake activity by 50%; removal of the C-terminal residue abolished all siRNA cellular uptake activity. These data indicate that the C-terminal region of the exemplary polynucleotide delivery-enhancing polypeptide PN73 is essential for the nucleotide cell uptake activity of the peptide.

peptide-siRNA conjugates enhance delivery of covalently linked transporters into cells:

the cellular uptake activity and MFI measurements of peptides of truncated forms of exemplary polynucleotide delivery-enhancing polypeptides suggest that these peptides may serve as delivery vehicles for a variety of molecular transporters. This involves covalently linking a desired effector molecule, including nucleic acids and peptides, to full-length PN73 or derivatives thereof. Defined delivery of effector molecules to specific cell types and/or intracellular organelles can be achieved by modifying the delivery peptide with specific moieties (lipid, peptide and/or carbohydrate groups).

Deletion analysis of exemplary polynucleotide delivery-enhancing polypeptides indicates that its N-terminus is critical for the ability of the peptide to bind and deliver nucleic acids (e.g., siNAs) into cells. However, even in the case of N-terminal deletions, these non-nucleotide-binding and severely attenuated nucleotide-binding peptides retain their membrane adhesion and fusion domains (e.g., PN 361; PN 735; PN 655; PN 654; PN 653; PN652 and PN651 and derivatives thereof). Given that these peptides are unable to bind nucleotides, these peptides can still be used to deliver siNA into cells by covalently linking the siNA to the membrane adhesion and/or fusion domain of the peptide. Thus, the siNA covalent bond remedies the defect that the peptide cannot bind nucleotides and allows for efficient peptide-mediated delivery of siNA into cells. Furthermore, siNA/peptide conjugates need not be limited to truncated forms of the exemplary polynucleotide delivery-enhancing polypeptides of choice, and may also include the full length of PN73 and derivatives thereof.

The following are non-limiting examples of methods for generating a siNA/peptide covalent bond. The peptide and siRNA molecule must incorporate functional groups with specific moieties to allow covalent binding to each other. For peptides, a functional group is added to the N-terminus, for example, with 3-maleimidopropionic acid. However, other functional groups such as bromo or iodoacetoxy are known to work as well. For RNA molecules, the 5 'end of the sense strand or the 3' end of the antisense strand is added with a functional group, for example, with a 1-O-dimethoxytrityl-hexyl-disulfide linker, according to the following synthesis method.

The 5' modified siNA was reacted with 0.393mg (3eq) of tris (2-carboxyethyl) phosphine (TCEP) in 0.3ml of 0.1M triethylamine acetate (TEAA) buffer (pH 7.0) at room temperature for 3h to reduce the free thiol group. Reduced oligonucleotides inMS C184.6X 50mm column, with a linear gradient CH of 0 to 30% in 0.1M TEAA buffer (pH 7)₃CN elution 20 min (t)_r5.931min), purified by RP HPLC.

The above-described conjugates were prepared. Purified reduced siNA (1.361mg, 0.19085 μmol) was dissolved in 0.2ml of 0.1M TEAA buffer (pH 7) and the peptide bearing the maleimide moiety was then conjugated to the N-terminus of the peptide (0.79mg, 1.5eq) and added to the siNA solution. After addition of the peptide, 150. mu.l of 75% CH was added₃The precipitate was dissolved by CN/0.1M TEAA. After stirring overnight at room temperature, the resulting conjugate was purified by RP HPLC atMS C184.6X 50mm column, with a linear gradient CH of 0 to 30% in 0.1M TEAA buffer (pH 7)₃CN eluted 20 min, the next 5 min (t)_r21.007min) was eluted with 100% C. The amount of conjugate was determined spectrophotometrically, based on the molar absorption coefficient calculated at λ -260 nm. MALDI mass spectrometry analysis showed that the peak of the observed conjugate (10585.3amu) matched the calculated mass.

The sense strand of the peptide conjugate and the attached antisense strand were annealed by heating at 90 ℃ for 2 minutes and then incubating at 37 ℃ for 1 hour in 50mM potassium acetate, 1mM magnesium acetate and 15mM HEPES, pH 7.4. The formation of double stranded RNA conjugates was confirmed by non-denaturing (15%) polyacrylamide gel electrophoresis followed by ethidium bromide staining.

The results of the cellular uptake activity are shown in table 25 below.

Table 25:

percentage of cells that take up siRNA by peptide-siRNA conjugates

The results in table 25 show that PN651-siRNA conjugate enhances siRNA uptake into cells. Then, MFI was measured, and the results thereof are presented in table 26.

Table 26:

relative amount of Cy5-siRNA delivered by peptide-siRNA conjugate (MFI)

The MFI results shown in table 26 are consistent with the data for percent siRNA uptake shown in table 25. These data indicate that PN651-siRNA conjugates enhance uptake of siRNA into cells.

Selected exemplary polynucleotide delivery enhances gene target knockdown activity of truncated forms of the polypeptide:

this example demonstrates that the siRNA/polynucleotide delivery-enhancing polypeptide complexes of the invention effectively knock down target gene expression. In particular, the ability of siRNA/polynucleotide delivery enhancing complexes to modulate the expression of human tumor necrosis factor-alpha (hTNF-alpha) gene was evaluated. The significance of targeting the hTNF- α gene is that it mediates the development and progression of Rheumatoid Arthritis (RA) when overexpressed in human and other mammalian subjects.

Using human monocytes as a model system, the effect of siRNA/polynucleotide delivery enhancing complex on hTNF- α gene expression was determined. Qneg represents random siRNA sequences as negative control. The observed Qneg knockdown activity was corrected to 100% (100% gene expression level), and the knockdown activity of each of the following siRNAs a19S21, 21/21, and LC20 was expressed as a relative percentage of negative controls. A19S21, 21/21 and LC20 are siRNAs targeting human TNF- α mRNA. Exemplary polynucleotide delivery enhancing polypeptides PN643 (full-length PN73 without C-terminal labeling), PN690 (full-length PN73 with C-terminal FITC labeling) and truncated forms of PN73 from the deletion series, PN660, PN735, PN654 and PN708 complexed with A19S21, 21/21 and LC20siRNAs to determine their effect on the ability of each siRNA to reduce the expression level of human monocyte hTNF-alpha gene

The experiments were performed as follows: human monocytes were plated at 100K/well/100. mu.l OptiMEM medium (Invitrogen) in 96-well flat bottom plates. Exemplary polynucleotide delivery-enhancing polypeptides were mixed with 20nM siRNA at a molar ratio of 1: 5 in OptiMEM medium at room temperature for 5 minutes. At the end of the incubation, FBS (final concentration 3%) was added to the mixture and 50. mu.l of this mixture was added to the cells. Cells were incubated at 37 ℃ for 3 hours. After incubation, cells were transferred to a V-plate and pelleted at 1500rpm for 5 minutes. The cells were resuspended in growth medium (IMDM with glutamate, non-essential amino acids and penicillin-streptomycin). After overnight incubation, monocytes were stimulated with 1ng/ml LPS (Sigma) for 3 hours to increase TNF-. alpha.expression levels. After LPS induction, cells were harvested as described above for mRNA quantification and supernatant was retained for protein quantification if necessary.

mRNA was determined using Genospectra's branched DNA technique according to the manufacturer's instructions. To quantify the mRNA levels in the cells, mRNA for the housekeeping gene (cypB) and the target gene (TNF-. alpha.) were determined and TNF-. alpha.readings were corrected for cypB to obtain relative luminescence units.

The knockdown activity of full-length and truncated forms of the exemplary polynucleotide delivery enhancing polypeptide PN73 are summarized in table 24 above. The "+" in the column "knockdown activity" indicates that the knockdown activity of the peptide/siRNA complex is 80% of that of the Qneg negative control siRNA (20% reduction in mRNA levels compared to the Qneg negative control). "+/-" indicates that the knockdown activity of the peptide/siRNA complex was about 90% of the Qneg negative control siRNA (10% reduction in mRNA levels compared to the Qneg negative control). Finally, "-" indicates that the peptide/siRNA complex had no significant knockdown activity compared to the Qneg negative control.

In summary, PN643 (full length PN73 without FITC labeling) and PN690 (full length PN73 with FITC labeling) had the same siRNA knockdown activity against all siRNAs tested, indicated by "+" in the "knockdown activity" column (results shown in table 24). In addition, PN660 had similar siRNA knockdown activity to all siRNAs tested as PN643 and PN690, suggesting that the terminal 9 residues at the N-terminus of the PN73 peptide did not affect the siRNAs-mediated targeted TNF- α mRNA knockdown activity. PN654 showed moderate knockdown activity for a19S21 and 21/21siRNAs, but not for LC20siRNA (knockdown activity is shown as "±) in the knockdown activity bar. However, siRNAs complexed with PN708 or PN735 did not result in observable knockdown activity for either of the siRNAs.

These results indicate that the truncated forms of the exemplary polynucleotide delivery-enhancing polypeptide PN73, specifically PN660 and PN654, do not interfere with the ability of siRNAs to reduce target gene mRNA levels, providing a novel approach to improving the delivery of therapeutic siRNAs for the treatment of human diseases such as RA.

Example 19

Features of the polynucleotide delivery-enhancing polypeptide PN708

This example further explores siRNA cellular uptake activity, MFI measurement and knockdown activity of siRNAs complexed with PN708 peptide. The peptides available are listed in the PN73 deletion series (see table 23 in example 17).

As described above, cellular uptake analysis determined the number of cells that received siRNA coupled to Cy5 when complexed with the peptide. Flow cytometry was used to assess cellular uptake of siRNA. Percent uptake was calculated from the number of cells containing the Cy 5-coupled siRNA divided by the total number of transfected and untransfected cultured cells. The Mean Fluorescence Intensity (MFI) was measured by flow cytometry and the number of siRNA coupled to Cy5 found inside the cells was determined. The MFI value is directly related to the amount of intracellular Cy5 coupled siRNA, thus, a higher MFI value indicates a greater amount of intracellular Cy5 coupled siRNA.

In this example, a greater range of peptide concentrations than in the previous examples was used to determine siRNA cellular uptake activity and efficacy of MIF measurements. Furthermore, cell viability was assessed. In this example, exemplary polynucleotide delivery enhancing polypeptides PN643 (full length PN73 unlabeled at the C-terminus), PN690 (full length PN73 labeled with FITC at the C-terminus), and PN708 (15 polynucleotides resulting from the deletion of 21 residues from the PN 73N-terminus) were tested at 5. mu.M, 10. mu.M, 20. mu.M, and 40. mu.M. Also detected were PN643 and PN690 of 2.5. mu.M, and PN690 of 1.25. mu.M. 80 μ M of PN643 and PN708 are also detected.

As shown in table 27 below, the non-FTTC labeled PN73(PN643) peptide achieved nearly 100% siRNA uptake at a concentration of 10 μ M. However, when PN73 peptide was labeled with FITC label, its maximal cellular uptake activity decreased to about 70%. PN708 showed a dose-dependent increase in siRNA cellular uptake activity. PN708 reached a maximum cellular uptake activity of 95% at 80 μ M. For the full-length PN73 peptide, cell viability decreased with increasing peptide concentration. In contrast, cells incubated with PN708 peptide at all concentrations tested maintained over 90% cell viability. The Cy5-MFI measurement further showed that the truncated peptide PN708 delivered approximately twice the amount of Cy5-siRNA into the cells as the full-length PN73(PN690) peptide.

Table 27:

summary of siRNA delivery enhancement features of PN708

These results show that the truncated exemplary polynucleotide delivery-enhancing polypeptide PN768(H2B residues 34-38) has the ability to increase the efficient delivery of siRNA into cells without adversely affecting cell viability.

It is further characterized by the determination of the effect of the truncated exemplary polynucleotide delivery-enhancing polypeptide PN708 on siRNA-mediated reduction of target gene expression. In this section of this example, the C-terminal FITC-tag of PN708 peptide was removed prior to assessing the ability of PN708 peptide in combination with siRNA to enhance target gene expression. Without FITC-labeling, this truncated exemplary polynucleotide delivery-enhancing peptide was named PN766 (see table 23 in example 17). The ability of the siRNA/peptide complexes to modulate the expression of the human tumor necrosis factor-alpha (hTNF-alpha) gene was evaluated. In this example, random siRNA sequences, Qneg, were used as negative controls to target hTNF- α mRNA in human monocytes using siRNAs LC20 and LC 17. The molar ratio of siRNA to peptide tested was 1: 5; 1: 10; 1: 25; 1: 50; 1: 75 and 1: 100. The concentrations of LC20 and LC17 used were both 20 nM.

Knockdown results showed that both LC20/PN766 and LC17/PN766 siRNA/peptide complexes at 1: 5, 1: 10, and 1: 25 reduced hTNF- α mRNA levels to about 70% -80% of the Qneg siRNA negative control (i.e., mRNA levels were reduced by 20% -30% compared to the Qneg negative control). siRNA/peptide ratios of 1: 50, 1: 75 and 1: 100 had no significant effect on hTNF- α mRNA levels compared to Qneg controls. No cytotoxic effect of human monocytes was observed in the presence of PN766 peptide.

These data indicate that the truncated exemplary polynucleotide delivery-enhancing polypeptide PN766 significantly reduces target gene mRNA levels when complexed with siRNA, suggesting that PN766 is an ideal siRNA delivery peptide for therapeutic siRNAs when treating RA in a mammalian subject.

Example 20

Exemplary polynucleotidesAmino acid substitutions and deletions within acid delivery-enhancing polypeptides do not affect peptide mediation siRNA cell uptake Activity of

This example demonstrates that the mutant exemplary polynucleotide delivery-enhancing polypeptides resulting from residue substitutions and/or deletions listed in table 28 below do not affect siRNA cellular uptake activity or MFI measurements as compared to the unmodified exemplary polynucleotide delivery-enhancing polypeptides. Table 28 below shows the residue substitutions that occurred within the exemplary polynucleotide delivery-enhancing polypeptide PN 73. Amino acids in the grayscale highlighting region represent unmodified, substituted, and/or deleted residues. The grayscale highlighting facilitates identification and comparison of unmodified residues within the PN73 peptide with substituted and/or deleted residues within the mutated exemplary polynucleotide delivery-enhancing polypeptides PN644, PN645, PN646, PN647, and PN 729. Exemplary polynucleotide delivery-enhancing polypeptides for mutations in the substituted bases are shown in bold and underlined. In addition, the symbol "^" in bold underline indicates one missing base within PN 729.

The purpose of exemplary polynucleotide delivery-enhancing polypeptides mutated by base substitutions or deletions was to assess the effect of these modifications on siRNA cellular uptake activity and efficiency of entry of siRNAs into cells.

Table 28:

PN73 residue substitution and deletion series

XRepresents a substituted amino acid;∧represents a deleted amino acid

Exemplary polynucleotide delivery-enhancing polypeptide PN73 specific residue substitutions and/or deletions include altering or increasing the number of aromatic amino acids and/or decreasing the number of negatively charged amino acids. Amino acids with aromatic functional groups (e.g., phenylalanine, tyrosine, tryptophan, and derivatives thereof) typically exist in the transmembrane domain of proteins due to their relatively non-polar (hydrophobic) character and the cell membrane penetrating ability of the facilitator protein. Negatively charged amino acids repel negatively charged nucleic acid phosphodiester backbone, thus disrupting the ability of the protein to bind nucleic acids. Thus, the rationale for aromatic amino acid substitutions within the PN73 peptide includes enhancing the cell penetrating function of the peptide and/or removing negatively charged amino acids to enhance nucleic acid binding of the peptide. The nucleic acid binding capacity of the peptide may also be enhanced by simply deleting negatively charged amino acids or substituting positively or neutral amino acids for negatively charged amino acids.

siRNA cellular uptake analysis and MFI measurement were done as previously described. The data are summarized in table 29 below. Each peptide was tested at concentrations of 0.63. mu.M, 1.25. mu.M, 2.5. mu.M and 5. mu.M. These results show that despite the presence of residue substitutions and/or deletions within the exemplified polynucleotide delivery-enhancing polypeptide PN73, these mutant peptides remained identical to the siRNA cellular uptake activity and MFI measurements of unmodified PN 73. These data indicate that residue substitutions and/or deletions do not affect the nucleic acid binding and cell membrane penetrating ability of the peptides. Moreover, the substituted and/or deleted residues within the exemplary polynucleotide delivery-enhancing polypeptides do not affect cell viability.

Table 29:

characterization summary of PN73 mutant mediated siRNA delivery

These results show that modifications of exemplary polynucleotide delivery-enhancing polypeptides, such as those produced by amino acid substitutions or deletions, or combinations thereof, deliver siRNA to a high percentage of cells with high efficiency.

Example 21

Enhancement of polynucleotide deliveryPolypeptide-mediated siRNA cell uptake activity

This example illustrates the polynucleotide delivery enhancing polypeptide-mediated siRNA cellular uptake activity listed in table 30 complexed with siRNA. Table 31 summarizes siRNA cell uptake data, Mean Fluorescence Intensity (MFI) measurements, and cell viability data for each polypeptide. Polypeptides that achieved 75% or greater percent siRNA cellular uptake are highlighted in gray scale in the "treatment" column. The specific percentage of cellular uptake of each corresponding siRNA of these highlighted siRNA/peptide complexes is also highlighted in gray scale in the column "percentage of cellular uptake of siRNA".

Table 30:

delivery-enhancing polypeptides screened based on siRNA cell uptake activity

Peptide ID #	SEQ IDNO：	Amino acid sequence	Name (R)
				PN680	178	RSVCRQIKICRRRGGCYYKCTNRPY-carboxamides	Androctonin
PN665	179	GFFALIPKIISSPLFKTLLSAVGSALSSSGDQE-carboxamides	Paradaxin
				PN734	180	GTAMRILGGVIPRKKRRQRRRPPQ-carboxamides	m-Calpain+TAT
PN681	181	KKKKKRFSFKKSFKLSGFSFKKNKK-carboxamides	MARCKS
				PN694	182	RQIKIWFQNRRMKWKK-amide	Cell-penetrating peptide
PN714	183	RQIRIWFQNRRMRWRR-carboxamides	PenArg
				PN760	184	RKKRRQRRRPPVAYISRGGVSTYYSDTVKGRFTRQKYNKRA-carboxamides	TAT + peptide P3a
PN759	185	LGLLLRHLRHHSNLLANIPRKKRRQRRRPP-	Egg binding protein + TAT
				PN682	186	KETWWETWWTEWSQPKKKRKV-AMIDE	Pep-1

The siRNA cellular uptake assay in this example determined the number of cells that received Cy 5-coupled LC20siRNA in the presence of peptide. LC20 is an oligonucleotide sequence for siRNA targeting human tumor necrosis factor-alpha (hTNF-alpha) mRNA. Cellular siRNA uptake was assessed by flow cytometry. Percent uptake is expressed as calculated from the number of cells containing the Cy 5-coupled siRNA divided by the total number of transfected and untransfected cultured cells. The Mean Fluorescence Intensity (MFI) was measured by flow cytometry and the number of siRNA coupled to Cy5 found inside the cells was determined. The MFI value is directly related to the amount of intracellular Cy5 coupled siRNA, thus, a higher MFI value indicates a greater amount of intracellular Cy5 coupled siRNA.

The following protocol was used to test the polynucleotide delivery-enhancing polypeptides listed in table 30. One day prior to transfection, Mouse Tail Fibroblast (MTF) cells were plated at approximately 80,000 per well in 24-well plates and cultured in complete medium. In the presence of 0.5. mu.M Cy 5-coupled siRNA, concentrations of 0.63. mu.M, 2.5. mu.M, 10. mu.M and 40. mu.M of each delivered peptide were detected, with the exception of the positive control. To form siRNA/peptide complexes, Cy 5-coupled siRNA and peptide were each diluted to twice the final concentration in Opti-MEM medium. The siRNA and peptide were mixed in equal volumes, complexed for 5 minutes at room temperature, and the siRNA/peptide complex was added to cells previously washed with Phosphate Buffered Saline (PBS). At 37 deg.C, 5% CO₂Cells were transfected for 3 hours under conditions. Cells were washed with PBS, treated with pancreatin, and then analyzed by flow cytometry. siRNA cellular uptake was determined by intracellular Cy5 fluorescence intensity. Cell viability was determined by propidium iodide uptake or annexin V-PE (BD biosciences) staining. To distinguish cellular uptake from membrane insertion of labeled siRNA (or fluorescently labeled peptide), the fluorescence used at the cell membrane surface was quenched with trypan blue. Trypan blue was added to the cells at a final concentration of 0.04%The sample is re-run on the flow cytometer to assess whether there is a change in fluorescence intensity that indicates that there is fluorescence localized on the membrane.

Table 31:

data from polypeptide-mediated siRNA delivery screen (NT as not detected)

As shown in table 31, column entitled "percent siRNA cell uptake", the negative control "no treatment" showed no siRNA cell uptake, while the positive control peptide reached a percent siRNA cell uptake of 95%. The Cy 5-coupled LC20siRNA complexed with the polynucleotide delivery-enhancing polypeptide PN680, PN681, PN709, PN760, PN759, or PN682 achieves a percentage of siRNA cellular uptake activity of greater than 75% or more. The polynucleotide delivery-enhancing polypeptides PN694 and PN714 exhibited moderate-intensity siRNA cellular uptake activity of 54% and 43%, respectively. In contrast, the polynucleotide delivery-enhancing polypeptides PN665 and PN734 did not show significant siRNA cellular uptake activity (less than 5%).

The characteristics of polynucleotide delivery that enhance the ability of polypeptides to transfect siRNAs into cells are further demonstrated by analysis of Mean Fluorescence Intensity (MFI). Cellular uptake was determined as the percentage of cells containing Cy 5-coupled siRNA, while MFI measurement determined the relative average amount of Cy 5-coupled siRNA that entered the cells. As shown in table 31, column entitled "siRNA Cy5 MFI," the siRNA coupled to Cy5 delivered through the positive control peptide PN643 reached an MFI of about 7 units. As expected, the "no treatment" negative control had no measurable MFI. The polynucleotide delivery-enhancing polypeptide PN665 was not detected with MFI. The MFI measurements for PN743, PN694 and PN714 are significantly lower than the MFI of the positive control. The polynucleotide delivery enhancing polypeptides PN680, PN709, and PN682 show similar MFI measurements as the PN643 positive control, while PN681 has twice the MFI of the positive control. Surprisingly, the MFI measurements for the polynucleotide delivery-enhancing polypeptides PN760 and PN759 were about 13-fold and 6-fold more, respectively, than the positive control MFI.

These data indicate that the polynucleotide delivery-enhancing polypeptide PN 680; PN 681; PN 709; PN 760; PN759 and PN682 complex with siRNA, effectively delivering siRNA into the cell.

Example 22

siRNAs for enhancing polypeptide transfection into cells by polynucleotide delivery Gene expression knockdown Activity of

This example demonstrates that a siRNA complexed with a polynucleotide delivery-enhancing polypeptide effectively knockdown mRNA expression of the siRNA target gene. In particular, the ability of the siRNA/peptide complexes to modulate the expression of the human tumor necrosis factor-alpha (hTNF-alpha) gene was evaluated. The significance of targeting the hTNF- α gene is that it mediates the development and progression of Rheumatoid Arthritis (RA) when overexpressed in human and other mammalian subjects.

The effect of siRNA/peptide complexes on hTNF- α gene expression was determined using human monocytes as a model system. Qneg represents random siRNA sequences as negative control. The observed Qneg knockdown activity was corrected to 100% (100% gene expression level), and the knockdown activity of each of the following siRNAs a19S21MD8, 21/21MD8, and LC20 was expressed as a relative percentage of negative controls. A19S21MD8, 21/21MD8 and LC20 are siRNAs targeting hTNF- α mRNA.

The polynucleotide delivery-enhancing polypeptide PN602 was the positive control for the acylated form used in the previous examples, both as a control for efficient delivery of siRNA into human monocytes and as a positive control for siRNA-mediated positive knockdown of the permissive knockdown activity of hTNF- α mRNA levels in this example. The polynucleotide delivery-enhancing polypeptides PN680 and PN681 were complexed with the siRNAs listed above to determine their effect on the ability of each siRNA to reduce the level of hTNF- α gene expression in human monocytes. Table 32 below summarizes the knockdown activity of all three polynucleotide delivery-enhancing polypeptides. The "+" in the column "knockdown activity" indicates that the knockdown activity of the peptide/siRNA complex is 80% of that of the Qneg negative control siRNA (20% reduction in mRNA levels compared to the Qneg negative control). "+/-" indicates that the knockdown activity of the peptide/siRNA complex was about 90% of the Qneg negative control siRNA (10% reduction in mRNA levels compared to the Qneg negative control). Finally, "-" indicates that the peptide/siRNA complex had no significant knockdown activity compared to the Qneg negative control.

Transfection of human monocytes in this example was performed according to the protocol described above.

mRNA was determined using the branched DNA technique (CA) of Genospectra (CA) according to the manufacturer's instructions. To quantify the mRNA levels in cells, mRNA for the housekeeping gene (cypB) and the target gene (TNF-. alpha.) were determined and TNF-. alpha.readings were corrected for cypB to obtain relative luminescence units.

Table 32:

SiRNAs with polynucleotide delivery enhancing polypeptide complexed siRNAs siRNA knockdown activity

The results shown in Table 32 indicate that all three siRNAs complexed with the positive control PN602 polynucleotide delivery-enhancing polypeptide in a ratio of 1: 5 and 1: 10 moderately reduced the level of hTNF- α gene expression compared to the Qneg negative control complexed with the same polypeptide. However, the same siRNAs complexed with the polynucleotide delivery-enhancing polypeptide PN681 in a ratio of 1: 5 and 1: 10 showed little or no knockdown activity relative to the Qneg negative control siRNA/PN681 complex. In contrast, polynucleotide delivery-enhancing polypeptide PN680 complexed with any hTNF- α specific siRNAs at a 1: 5 ratio demonstrated significant hTNF- α mRNA knock-down activity relative to the Qneg/PN680 control complex. Furthermore, the 1: 10 ratio of LC20/PN680 complex also showed significant knockdown activity compared to the Qneg/PN680 control complex.

These data indicate that the polynucleotide delivery-enhancing polypeptide PN680 delivers siRNAs into cells and allows for efficient siRNA-mediated gene silencing.

Although the foregoing invention has been described in some detail by way of example for purposes of clarity of understanding, it will be apparent to those 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 rather than of limitation. Various publications and other references are incorporated herein by reference in the foregoing disclosure for the sake of brevity. Each of these references is incorporated by reference herein in its entirety for all purposes. It is noted, however, that the various publications discussed herein are incorporated by reference only because their disclosure is prior to the filing date of the present application and the inventors reserve the right to precede these disclosures in view of prior inventions.

Sequence listing

<110>NASTECH PHARMACEUTICAL COMPANY，INC.

<120> pharmaceutical composition for delivering ribonucleic acid to cells

<130>04-03CIP-PCT

<140>

<141>

<150>11/223,699

<151>2005-09-08

<150>60/727,216

<151>2005-10-14

<150>60/733,664

<151>2005-11-04

<160>188

<170>PatentIn Ver.3.3

<210>1

<211>7

<212>PRT

<213> human immunodeficiency virus

<400>1

Lys Arg Arg Gln Arg Arg Arg

1 5

<210>2

<211>16

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: unknown penetrating PTD peptides

<400>2

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210>3

<211>34

<212>PRT

<213> herpes simplex virus

<400>3

Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr

1 5 10 15

Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro

20 25 30

Val Asp

<210>4

<211>16

<212>PRT

<213> human

<400>4

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

<210>5

<211>16

<212>PRT

<213> human

<400>5

Ala Ala Val Leu Leu Pro Val Leu Leu Pro Val Leu Leu Ala Ala Pro

1 5 10 15

<210>6

<211>15

<212>PRT

<213> human

<400>6

Val Thr Val Leu Ala Leu Gly Ala Leu Ala Gly Val Gly Val Gly

1 5 10 15

<210>7

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: gP41 fusion peptide

<400>7

Gly Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly

1 5 10 15

Ala

<210>8

<211>17

<212>PRT

<213> Mizhongmeikai crocodile

<400>8

Met Gly Leu Gly Leu His Leu Leu Val Leu Ala Ala Ala Leu Gln Gly

1 5 10 15

Ala

<210>9

<211>24

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: hCT derived peptide

<400>9

Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln

1 5 10 15

Thr Ala Ile Gly Val Gly Ala Pro

20

<210>10

<211>26

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: unknown delivery peptides

<400>10

Gly Trp Thr L6u Asn Ser Ala Gly Tyr Leu Leu Lys Ile Asn Leu Lys

1 5 10 15

Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu

20 25

<210>11

<211>16

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: unknown oligomer peptides

<400>11

Thr Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro Lys Lys Lys Lys

1 5 10 15

<210>12

<211>7

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: unknown arginine peptides

<400>12

Arg Arg Arg Arg Arg Arg Arg

1 5

<210>13

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: ampholytic model peptides

<400>13

Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys

1 5 10 15

Leu Ala

<210>14

<211>16

<212>PRT

<213> influenza virus

<400>14

Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

<210>15

<211>16

<212>PRT

<213> Sendai Virus

<400>15

Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly Val Ala Thr Ala

1 5 10 15

<210>16

<211>16

<212>PRT

<213> respiratory syncytial virus

<400>16

Phe Leu Gly Phe Leu Leu Gly Val Gly Ser AlaIle Ala Ser Gly Val

1 5 10 15

<210>17

<211>16

<212>PRT

<213> human immunodeficiency virus

<400>17

Gly Val Phe Val Leu Gly Phe Leu Gly Phe Leu Ala Thr Ala Gly Ser

1 5 10 15

<210>18

<211>16

<212>PRT

<213> Epulara virus

<400>18

Gly Ala Ala Ile Gly Leu Ala Trp Ile Pro Tyr Phe Gly Pro Ala Ala

1 5 10 15

<210>19

<211>56

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Zinc finger motifs

<400>19

Ala Cys Thr Cys Pro Tyr Cys Lys Asp Ser Glu Gly Arg Gly Ser Gly

1 5 10 15

Asp Pro Gly Lys Lys Lys Gln His Ile Cys His Ile Gln Gly Cys Gly

20 25 30

Lys Val Tyr Gly Lys Thr Ser His Leu Arg Ala His Leu Arg Trp His

35 40 45

Thr Gly Glu Arg Pro Phe Met Cys

50 55

<210>20

<211>54

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Zinc finger motifs

<400>20

Ala Cys Thr Cys Pro Asn Cys Lys Asp Gly Glu Lys Arg Ser Gly Glu

1 5 10 15

Gln Gly Lys Lys Lys His Val Cys His Ile Pro Asp Cys Gly Lys Thr

20 25 30

Phe Arg Lys Thr Ser Leu Leu Arg Ala His Val Arg Leu His Thr Gly

35 40 45

Glu Arg Pro Phe Val Cys

50

<210>21

<211>55

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Zinc finger motifs

<400>21

Ala Cys Thr Cys Pro Asn Cys Lys Glu Gly Gly Gly Arg Gly Thr Asn

1 5 10 15

Leu Gly Lys Lys Lys Gln His Ile Cys His Ile Pro Gly Cys Gly Lys

20 25 30

Val Tyr Gly Lys Thr Ser His Leu Arg Ala His Leu Arg Trp His Ser

35 40 45

Gly Glu Arg Pro Phe Val Cys

50 55

<210>22

<211>56

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Zinc finger motifs

<400>22

Ala Cys Ser Cys Pro Asn Cys Arg Glu Gly Glu Gly Arg Gly Ser Asn

1 5 10 15

Glu Pro Gly Lys Lys Lys Gln His Ile Cys His Ile Glu Gly Cys Gly

20 25 30

Lys Val Tyr Gly Lys Thr Ser His Leu Arg Ala His Leu Arg Trp His

35 40 45

Thr Gly Glu Arg Pro Phe Ile Cys

50 55

<210>23

<211>60

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Zinc finger motifs

<400>23

Arg Cys Thr Cys Pro Asn Cys Thr Asn Glu Met Ser Gly Leu Pro Pro

1 5 10 15

Ile Val Gly Pro Asp Glu Arg Gly Arg Lys Gln His Ile Cys His Ile

20 25 30

Pro Gly Cys Glu Arg Leu Tyr Gly Lys Ala Ser His Leu Lys Thr His

35 40 45

Leu Arg Trp His Thr Gly Glu Arg Pro Phe Leu Cys

50 55 60

<210>24

<211>58

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Zinc finger motifs

<400>24

Thr Cys Asp Cys Pro Asn Cys Gln Glu Ala Glu Arg Leu Gly Pro Ala

1 5 10 15

Gly Val His Ile Arg Lys Lys Asn Ile His Ser Cys His Ile Pro Gly

20 25 30

Cys Gly Lys Val Tyr Gly Lys Thr Ser His Leu Lys Ala His Leu Arg

35 40 45

Trp His Thr Gly Glu Arg Pro Phe Val Cys

50 55

<210>25

<211>53

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Zinc finger motifs

<400>25

Arg Cys Thr Cys Pro Asn Cys Lys Ala Ile Lys His Gly Asp Arg Gly

1 5 10 15

Ser Gln His Thr His Leu Cys Ser Val Pro Gly Cys Gly Lys Thr Tyr

20 25 30

LysLys Thr Ser His Leu Arg Ala His Leu Arg Lys His Thr Gly Asp

35 40 45

Arg Pro Phe Val Cys

50

<210>26

<211>56

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Zinc finger motifs

<400>26

Pro Gln Ile Ser Leu Lys Lys Lys Ile Phe Phe Phe Ile Phe Ser Asn

1 5 10 15

Phe Arg Gly Asp Gly Lys Ser Arg Ile His Ile Cys His Leu Cys Asn

20 25 30

Lys Thr Tyr Gly Lys Thr Ser His Leu Arg Ala His Leu Arg Gly His

35 40 45

Ala Gly Asn Lys Pro Phe Ala Cys

50 55

<210>27

<211>31

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Polypeptides

<400>27

Trp Trp Glu Thr Trp Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn

1 5 10 15

Phe Ser Thr Arg Gln Ala Arg Arg Asn His Arg Arg Arg His Arg

20 25 30

<210>28

<211>16

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Polypeptides

<400>28

Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu

1 5 10 15

<210>29

<211>16

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Polypeptides

<400>29

Arg Val Ile Arg Val Trp Phe Gln Asn Lys Arg Cys Lys Asp Lys Lys

1 5 10 15

<210>30

<211>39

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Polypeptides

<400>30

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Arg Lys

1 5 10 15

Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Arg Lys Lys Arg Arg

20 25 30

Gln Arg Arg Arg Pro Pro Gln

35

<210>31

<211>22

<212>PRT

<213> unknown organism

<220>

<223> description of unknown organisms: exemplary Polypeptides

<400>31

Gly Glu Gln Ile Ala Gln Leu Ile Ala Gly Tyr Ile Asp Ile Ile Leu

1 5 10 15

Lys Lys Lys Lys Ser Lys

20

<210>32

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of combined DNA/RNA molecules: synthesis of siRNA

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>32

cuacacaaau cagcgauuutt 21

<210>33

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of combined DNA/RNA molecules: synthesis of siRNA

<220>

<223> unknown biological description: synthesis of siRNA

<400>33

aaaucgcuga uuuguguagt t 21

<210>34

<211>22

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>34

gcaagcugac ccugaaguuc au 22

<210>35

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>35

Arg Arg Arg Gln Arg Arg Lys Arg Gly Gly Asp Ile Met Gly Glu Trp

1 5 10 15

Gly Asn Glu Ile Phe Gly Ala Ile Ala Gly Phe Leu Gly

20 25

<210>36

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>36

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Cys

1 5 10

<210>37

<211>28

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>37

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln

20 25

<210>38

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>38

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Cys

20 25

<210>39

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>39

Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Cys Ala Ala Val

1 5 10 15

Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

20 25

<210>40

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>40

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln

1 5 10

<210>41

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>41

Arg Arg Arg Gln Arg Arg Lys Arg Gly Gly Asp Ile Met Gly Glu Trp

1 5 10 15

Gly Asn Glu Ile Phe Gly Ala Ile Ala Gly Phe Leu Gly

20 25

<210>42

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>42

Cys Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Tyr Gly Arg

1 5 10 15

Lys Lys Arg Arg Gln Arg Arg Arg Gly

20 25

<210>43

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>43

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln

1 5 10

<210>44

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>44

Lys Leu Trp Lys Ala Trp Pro Lys Leu Trp Lys Lys Leu Trp Lys Pro

1 5 10 15

<210>45

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>45

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

Arg Arg Arg Arg Arg Arg

20

<210>46

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>46

Arg Leu Trp Arg Ala Leu Pro Arg Val Leu Arg Arg Leu Leu Arg Pro

1 5 10 15

<210>47

<211>28

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>47

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

Ser Gly Ala Ser Gly Leu Asp Lys Arg Asp Tyr Val

20 25

<210>48

<211>28

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>48

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

Ser Gly Ala Ser Gly Leu Asp Lys Arg Asp Tyr Val

20 25

<210>49

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>49

Ser Gly Ala Ser Gly Leu Asp Lys Arg Asp Tyr Val Ala Ala Val Ala

1 5 10 15

Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

20 25

<210>50

<211>33

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>50

Leu Leu Glu Thr Leu Leu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg

1 5 10 15

Asn Phe Ser Thr Arg Gln Ala Arg Arg Asn His Arg Arg Arg His Arg

20 25 30

Arg

<210>51

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>51

Ala Ala Val Ala Cys Arg Ile Cys Met Arg Asn Phe Ser Thr Arg Gln

1 5 10 15

Ala Arg Arg Asn His Arg Arg Arg His Arg Arg

20 25

<210>52

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>52

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210>53

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>53

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210>54

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>54

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

Asp Ile Met Gly Glu Trp Gly Asn Glu Ile Phe Gly Ala Ile Ala Gly

20 25 30

Phe Leu Gly

35

<210>55

<211>37

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>55

Ser Gly Arg Gly Lys Gln Gly Gly Lys Ala Arg Ala Lys Ala Lys Thr

1 5 10 15

Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg

20 25 30

Leu Leu Arg Lys Gly

35

<210>56

<211>38

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>56

Ser Gly Arg Gly Lys Gln Gly Gly Lys Ala Arg Ala Lys Ala Lys Thr

1 5 10 15

Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg

20 25 30

Leu Leu Arg Lys Gly Cys

35

<210>57

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>57

Lys Gly ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys

20

<210>58

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>58

Lys Lys Asp Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser

1 5 10 15

Val Tyr Val Tyr Lys Val Leu Lys Gln

20 25

<210>59

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>59

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>60

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>60

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu

1 5 10 15

Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu

20 25

<210>61

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>61

Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys

1 5 10 15

Leu Ala

<210>62

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>62

Lys G1u Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Arg Lys Val

20

<210>63

<211>28

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>63

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Gly

1 5 10 15

Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln

20 25

<210>64

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>64

Arg Arg Arg Arg Arg Arg Arg

1 5

<210>65

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>65

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

1 5 10

<210>66

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>66

Arg Arg Arg Gln Arg Arg Lys Arg Gly Gly Gln Gln Gln Gln Gln Gln

1 5 10 15

Gln Gln Gln Gln

20

<210>67

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>67

Arg Val Ile Arg Trp Phe Gln Asn Lys Arg Cys Lys Asp Lys Lys

1 5 10 15

<210>68

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>68

Leu Gly Leu Leu Leu Arg His Leu Arg His His Ser Asn Leu Leu Ala

1 5 10 15

Asn Ile

<210>69

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>69

Gly Gln Met Ser Glu Ile Glu Ala Lys Val Arg Thr Val Lys Leu Ala

1 5 10 15

Arg Ser

<210>70

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>70

Lys Leu Trp Ser Ala Trp Pro Ser Leu Trp Ser Ser Leu Trp Lys Pro

1 5 10 15

<210>71

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>71

Lys Lys Lys Lys Lys Lys Lys Lys Lys

1 5

<210>72

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>72

Ala Ala Arg Leu His Arg Phe Lys Asn Lys Gly Lys Asp Ser Thr Glu

1 5 10 15

Met Arg Arg Arg Arg

20

<210>73

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>73

Gly Leu Gly Ser Leu Leu Lys Lys Ala Gly Lys Lys Leu Lys Gln Pro

1 5 10 15

Lys Ser Lys Arg Lys Val

20

<210>74

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD_RES

<222>(1)

<223> bis-methyl tyrosine

<400>74

Xaa Arg Phe Lys Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

1 5 10

<210>75

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>75

Trp Arg Phe Lys

1

<210>76

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>76

Trp Arg Phe Lys Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

1 5 10

<210>77

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>77

Tyr Arg Phe Lys

1

<210>78

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>78

Tyr Arg Phe Lys Tyr Arg Phe Lys Tyr Arg Phe Lys

1 5 10

<210>79

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>79

Trp Arg Phe Lys Lys Ser Lys Arg Lys Val

1 5 10

<210>80

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>80

Trp Arg Phe Lys Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala

1 5 10 15

Leu Leu Ala Pro

20

<210>81

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD_RES

<222>(1)

<223> bis-methyl tyrosine

<400>81

Xaa Arg Phe Lys

1

<210>82

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>82

Tyr Arg Phe Lys

1

<210>83

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD_RES

<222>(1)

<223> bis-methyl tyrosine

<400>83

Xaa Arg Phe Lys

1

<210>84

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>84

Trp Arg Phe Lys

1

<210>85

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD_RES

<222>(1)

<223> bis-methyl tyrosine

<400>85

Xaa Tyr Arg Trp Lys

1 5

<210>86

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD_RES

<222>(4)

<223> bis-methyl tyrosine

<400>86

Lys Phe Arg Xaa

1

<210>87

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>87

Trp Arg Phe Lys Trp Arg Phe Lys

1 5

<210>88

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>88

Trp Arg Phe Lys Trp Arg Phe Lys Trp Arg Phe Lys

1 5 10

<210>89

<211>28

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>89

Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Ala Ala Val Ala

1 5 10 15

Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

20 25

<210>90

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>90

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>91

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>91

Arg Gly Ser Arg Arg Ala Val Thr Arg Ala Gln Arg Arg Asp Gly Arg

1 5 10 15

Arg Arg Arg Arg Ser Arg Arg Glu Ser Tyr Ser Val Tyr Val Tyr Arg

20 25 30

Val Leu Arg Gln

35

<210>92

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>92

Arg Val Ile Arg Trp Phe Gln Asn Lys Arg Ser Lys Asp Lys Lys

1 5 10 15

<210>93

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>93

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu

1 5 10 15

Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu

20 25

<210>94

<211>28

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>94

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln

20 25

<210>95

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>95

Lys Glu Thr Typ Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Arg Lys Val

20

<210>96

<211>28

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>96

Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly

1 5 10 15

Ala Trp Ser Gln Pro Lys Ser Lys Arg Lys Val Cys

20 25

<210>97

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>97

Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln

1 5 10 15

<210>98

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>98

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>99

<211>33

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>99

Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys

1 5 10 15

Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys

20 25 30

Gln

<210>100

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>100

Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg Ser Arg

1 5 10 15

Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln

20 25 30

<210>101

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>101

Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser

1 5 10 15

Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln

20 25

<210>102

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>102

Arg Arg Arg Gln Arg Arg Lys Arg Gly Gly Asp Ile Met Gly Glu Trp

1 5 10 15

Gly Asn Glu Ile Phe Gly Ala Ile Ala Gly Phe Leu Gly

20 25

<210>103

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>103

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>104

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>104

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Arg Lys Val

20

<210>105

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>105

Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Cys Ala Ala Val

1 5 10 15

Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

20 25

<210>106

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>106

Trp Arg Phe Lys Cys

1 5

<210>107

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>107

Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys

1 5 10 15

Leu Ala

<210>108

<211>27

<212>PRT

<213> Artificial sequence

<223> description of artificial sequences: synthetic peptides

<400>108

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu

1 5 10 15

Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu

20 25

<210>109

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>109

gcguggagcu gagagauaa 19

<210>110

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>110

gccuguagcc cauguugua 19

<210>111

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>111

gguaugagcc caucuaucu 19

<210>112

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>112

ccagggaccu cucucuaau 19

<210>113

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>113

gcccgacuau cucgacuuu 19

<210>114

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>114

ugacaagccu guagcccau 19

<210>115

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>115

ggucuacuuu gggaucauu 19

<210>116

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>116

cccagggacc ucucucuaa 19

<210>117

<211>23

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>117

aaucggcccg acuaucucga cuu 23

<210>118

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>118

aauggcgugg agcugagaga u 21

<210>119

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>119

aaccuccucu cugccaucaa g 21

<210>120

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>120

aacugaaagc augauccggg a 21

<210>121

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>121

aaucucgacu uugccgaguc u 21

<210>122

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>122

aagggugacc gacucagcgc u 21

<210>123

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>123

aaucagccgc aucgccgucu c 21

<210>124

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>124

aacccaugug cuccucaccc a 21

<210>125

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>125

aagcuccagu ggcugaaccg c 21

<210>126

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>126

aagucagauc aucuucucga a 21

<210>127

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>127

aagggaccuc ucucuaauca g 21

<210>128

<211>23

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>128

ccucagccuc uucuccuucc uga 23

<210>129

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>129

aauccucagc cucuucuccu u 21

<210>130

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>130

aaccaaugcc cuccuggcca a 21

<210>131

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>131

cugauuaagu ugucuaaaca a 21

<210>132

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>132

ccgacucagc gcugagauca a 21

<210>133

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>133

cuugugauua uuuauuauuu a 21

<210>134

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>134

aagccuguag cccauguugu a 21

<210>135

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>135

uagggucgga acccaagcuu a 21

<210>136

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>136

cugaaagcau gauccggga 19

<210>137

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>137

aggcggugcu uguuccuca 19

<210>138

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>138

ccaccacgcu cuucugccu 19

<210>139

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>139

agggaccucu cucuaauca 19

<210>140

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>140

ugacaagccu guagcccau 19

<210>141

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>141

gccuguagcc cauguugua 19

<210>142

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>142

uagcccaugu uguagcaaa 19

<210>143

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>143

ccaaugcccu ccuggccaa 19

<210>144

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>144

ccaauggcgu ggagcugag 19

<210>145

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>145

ggcguggagc ugagagaua 19

<210>146

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>146

gcguggagcu gagagauaa 19

<210>147

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>147

gccuguaccu caucuacuc 19

<210>148

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>148

ccuccucucu gccaucaag 19

<210>149

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>149

gguaugagcc caucuaucu 19

<210>150

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>150

gcuggagaag ggugaccga 19

<210>151

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>151

gagaagggug accgacuca 19

<210>152

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>152

gcccgacuau cucgacuuu 19

<210>153

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>153

gcaggucuac uuugggauc 19

<210>154

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>154

ggucuacuuu gggaucauu 19

<210>155

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>155

ugggaucauu gcccuguga 19

<210>156

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>156

ggucggaacc caagcuuag 19

<210>157

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>157

ccagaaugcu gcaggacuu 19

<210>158

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>158

gagaagaccu caccuagaa 19

<210>159

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>159

gaagaccuca ccuagaaau 19

<210>160

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>160

ccagauguuu ccagacuuc 19

<210>161

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>161

cuauuuaugu uugcacuug 19

<210>162

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>162

ucuaaacaau gcugauuug 19

<210>163

<211>18

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>163

gaccaacugu cacucauu 18

<210>164

<211>125

<212>PRT

<213> human

<400>164

Met Pro Glu Pro Ala Lys Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys

1 5 10 15

Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Ser Lys Lys Arg Lys Arg

20 25 30

Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Val

35 40 45

His Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly Ile Met Asn Ser

50 55 60

Phe Val Asn Asp Ile Phe Glu Arg Ile Ala Gly Glu Ala Ser Arg Leu

65 70 75 80

Ala His Tyr Asn Lys Arg Ser Thr Ile Thr Ser Arg Glu Ile Gln Thr

85 90 95

Ala Val Arg Leu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val Ser

100 105 110

Glu Gly Thr Lys Ala Val Thr Lys Tyr Thr Ser Ser Lys

115 120 125

<210>165

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>165

Lys Asp Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val

1 5 10 15

Tyr Val Tyr Lys Val Leu Lys Gln

20

<210>166

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>166

Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr

1 5 10 15

Lys Val Leu Lys Gln

20

<210>167

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>167

Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu

1 5 10 15

Lys Gln

<210>168

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>168

Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln

1 5 10 15

<210>169

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>169

Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln

1 5 10

<210>170

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>170

Val Tyr Val Tyr Lys Val Leu Lys Gln

1 5

<210>171

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>171

Tyr Lys Val Leu Lys Gln

1 5

<210>172

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>172

Lys Val Leu Lys Gln

1 5

<210>173

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>173

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Trp Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>174

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>174

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Trp Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>175

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>175

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Phe Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>176

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>176

Lys Gly Ser Phe Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Phe Lys Phe Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>177

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>177

Lys Gly Ser Phe Lys Ala Val Thr Lys Ala Gln Lys Lys Phe Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Ser Phe Ser Val Tyr Val Tyr Lys Val

20 25 30

Leu Lys Gln

35

<210>178

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>178

Arg Ser Val Cys Arg Gln Ile Lys Ile Cys Arg Arg Arg Gly Gly Cys

1 5 10 15

Tyr Tyr Lys Cys Thr Asn Arg Pro Tyr

20 25

<210>179

<211>33

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>179

Gly Phe Phe Ala Leu Ile Pro Lys Ile Ile Ser Ser Pro Leu Phe Lys

1 5 10 15

Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Ser Gly Asp Gln

20 25 30

Glu

<210>180

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>180

Gly Thr Ala Met Arg Ile Leu Gly Gly Val Ile Pro Arg Lys Lys Arg

1 5 10 15

Arg Gln Arg Arg Arg Pro Pro Gln

20

<210>181

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>181

Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys Leu Ser

1 5 10 15

Gly Phe Ser Phe Lys Lys Asn Lys Lys

20 25

<210>182

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>182

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210>183

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>183

Arg Gln Ile Arg Ile Trp Phe Gln Asn Arg Arg Met Arg Trp Arg Arg

1 5 10 15

<210>184

<211>41

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>184

Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Val Ala Tyr Ile Ser

1 5 10 15

Arg Gly Gly Val Ser Thr Tyr Tyr Ser Asp Thr Val Lys Gly Arg Phe

20 25 30

Thr Arg Gln Lys Tyr Asn Lys Arg Ala

35 40

<210>185

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>185

Leu Gly Leu Leu Leu Arg His Leu Arg His His Ser Asn Leu Leu Ala

1 5 10 15

Asn Ile Pro Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro

20 25 30

<210>186

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>186

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Arg Lys Val

20

<210>187

<211>19

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of siRNA

<400>187

uagcccaugu uguagcaaa 19

<210>188

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides of the general formula

<220>

<221>MOD_RES

<222>(2)..(5)

<223> this region may contain 2 or 4 variable residues

<220>

<221>MOD_RES

<222>(7)..(18)

<223> variable residue

<220>

<221>MOD_RES

<222>(20)..(22)

<223> variable residue

<400>188

Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa His Xaa Xaa Xaa His

20

Claims (37)

1. A composition comprising a polynucleotide delivery-enhancing polypeptide and a double-stranded ribonucleic acid (dsRNA), wherein the polynucleotide delivery-enhancing polypeptide is amphiphilic and comprises nucleic acid binding properties.

2. The composition of claim 1, wherein the polynucleotide delivery-enhancing polypeptide comprises from about 5 to about 40 amino acids and comprises a sequence selected from the group consisting of poly (Lys, Tryp) 4: 1MW20,000-50,000, poly (Orn, Trp) 4: 1MW20,000-50,000, melittin, histone H1, histone H3 and histone H4, SEQ ID NOS: 27-31, 35-42, 45, 47, 50-59, 62, 63, 67, 68, 73, 74, 76, 78-87, 89-92, 94-108, 164, 178, and 180, 186.

3. The composition of claim 1, wherein the composition results in uptake of the dsRNA into an animal cell.

4. The composition of claim 3, wherein the animal cell is a mammalian cell.

5. 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, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is acetylated.

8. The composition of claim 1, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is pegylated.

9. The composition of claim 1, wherein the dsRNA is a small interfering ribonucleic acid (siRNA) consisting of about 10 to about 40 base pair sequence complementary to a portion of a tumor necrosis factor-alpha (TNF-a) gene.

10. The composition of claim 1, wherein the dsRNA is an siRNA consisting of a sequence selected from the group consisting of SEQ ID NOS: 109, 163, and 187 from about 10 to about 40 base pairs.

11. The composition of claim 1, wherein the polynucleotide delivery-enhancing polypeptide is mixed, complexed or conjugated to the dsRNA.

12. The composition of claim 1, wherein the polynucleotide delivery-enhancing polypeptide binds to the dsRNA.

13. 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-trimethylammonium chloride, 1, 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane, 1, 2-ditetradecyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2, 3-dioleoyloxy-N- [2 (spermine carboxamido) ethyl ] ammonium]ammonium-N, N-dimethyl-1-propyltrifluoroacetate, 1, 3-dioleoyloxy-2- (6-carboxyspermine) -propionamide, 5-carboxysperminamine aminoacetic acid dioctadecylamide, tetramethyltetrapalmitospermine, tetramethyltetraoleospermine, tetramethyltetralauryl spermine, tetramethyltetramyristyl spermine and tetramethyldioleospermine, DOTMA (N- [ 1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane), DMRIE (1, 2-bistetradecyloxypropyl-3-dimethylhydroxyethylammonium bromide), DDAB (dioctadecyldimethylammonium bromide), Polyvalent cation liposome, lipospermine, DOSPA (2, 3-dioleoyloxy-N- [2 (spermine carboxamide) ethyl)]ammonium-N, N-dimethyl-1-propyltrifluoroacetate), DOSPER (1, 3-dioleoyloxy-2- (6-carboxyspermine) -propionamide, di-and tetra-alkyl-tetra-methyl spermine), TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleoyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMTMTMTMTMTMS (tetramethyltetramyristyl spermine), TMDS (tetramethyldioleoyl spermine) DOGS (dioctadecylamidoglycyl spermine (C)) Cationic lipid compositions consisting of cationic lipid bound to non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol, a 3: 1(w/w) mixture of DOSPA and DOPE, and a 1: 1(w/w) mixture of DOTMA and DOPE.

15. A method of causing uptake of a double-stranded ribonucleic acid (dsRNA) into an animal cell, comprising incubating the animal cell with a mixture comprising a polynucleotide delivery-enhancing polypeptide and the dsRNA, wherein the polynucleotide delivery-enhancing polypeptide is amphipathic and comprises nucleic acid binding properties.

16. A method of modifying expression of a target gene in an animal cell comprising incubating the animal cell with a mixture comprising a polynucleotide delivery-enhancing polypeptide and a double-stranded ribonucleic acid (dsRNA), wherein the polynucleotide delivery-enhancing polypeptide is amphipathic and comprises nucleic acid binding properties, wherein the dsRNA is complementary to a region of the target gene.

17. The method of claim 15 or 16, wherein the animal cell is a mammalian cell.

18. A method of altering a phenotype of an animal subject comprising administering to the animal subject a mixture of a polynucleotide delivery-enhancing polypeptide and a double-stranded ribonucleic acid (dsRNA), wherein the polynucleotide delivery-enhancing polypeptide is amphipathic and comprises nucleic acid binding properties, 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 polynucleotide delivery-enhancing polypeptide comprises from about 5 to about 40 amino acids and comprises all or part of a sequence selected from the group consisting of poly (Lys, Tryp) 4: 1MW20,000-50,000, poly (Orn, Trp) 4: 120,000-50,000, melittin, histone H1, histone H3 and histone H4, SEQ ID NOS 27-31, 35-42, 45, 47, 50-59, 62, 63, 67, 68, 73, 74, 76, 78-87, 89-92, 94-108, 164-178 and 180-186.

21. The method of claim 15, 16, or 18, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is acetylated.

22. The method of claim 15, 16 or 18, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is pegylated.

23. The method of claim 15, 16 or 18, wherein the dsRNA is a small interfering ribonucleic acid (siRNA) consisting of about 10 to about 40 base pair sequence that is complementary to a portion of a tumor necrosis factor-alpha (TNF- α) gene.

24. The method of claim 15, 16 or 18 wherein the dsRNA is an siRNA consisting of about 10 to about 40 base pair sequence selected from the group consisting of SEQ ID NOS 109-163 and 187.

25. The method of claim 15, 16 or 18, wherein the polynucleotide delivery-enhancing polypeptide is mixed, complexed or conjugated to the dsRNA.

26. The method of claim 15, 16 or 18, wherein the polynucleotide delivery-enhancing polypeptide binds to the dsRNA.

27. The method of claim 15, 16 or 18, further comprising a cationic lipid.

28. The method of claim 27, wherein the cationic lipid is selected from the group consisting of N- [ 1- (2, 3-dioleoyloxy) propyl ] -N, N-trimethylammonium chloride, 1, 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane, 1, 2-ditetradecyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2, 3-dioleoyloxy-N- [2 (spermine carboxamido) ethyl ] ammonium]ammonium-N, N-dimethyl-1-propyltrifluoroacetate, 1, 3-dioleoyloxy-2- (6-carboxyspermine) -propionamide, 5-carboxysperminamine aminoacetic acid dioctadecylamide, tetramethyltetrapalmitospermine, tetramethyltetraoleospermine, tetramethyltetralauryl spermine, tetramethyltetramyristyl spermine and tetramethyldioleospermine, DOTMA (N- [ 1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane), DMRIE (1, 2-bistetradecyloxypropyl-3-dimethylhydroxyethylammonium bromide), DDAB (dioctadecyldimethylammonium bromide), Polyvalent cation liposome, lipospermine, DOSPA (2, 3-dioleoyloxy-N- [2 (spermine carboxamide) ethyl)]ammonium-N, N-dimethyl-1-propyltrifluoroacetate), DOSPER (1, 3-dioleoyloxy-2- (6-carboxyspermine) -propionamide, di-and tetra-alkyl-tetra-methyl spermine), TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleoyl spermine), TMTLS (tetramethyltetralauryl spermine), TMTMTMTMTMTMTMS (tetramethyltetramyristyl spermine), TMDS (tetramethyldioleoyl spermine) DOGS (dioctadecylamidoglycyl spermine (C)) Cationic lipid compositions consisting of cationic lipid bound to non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol, a 3: 1(w/w) mixture of DOSPA and DOPE, and a 1: 1(w/w) mixture of DOTMA and DOPE.

29. Use of a mixture comprising a polynucleotide delivery-enhancing polypeptide and a double-stranded ribonucleic acid (dsRNA) for the preparation of a medicament for treating a tumor necrosis factor-alpha (TNF- α) -related inflammatory condition in an animal subject, wherein said polynucleotide delivery-enhancing polypeptide is amphipathic and comprises nucleic acid binding properties, wherein said medicament is capable of reducing TNF- α RNA levels, thereby preventing or reducing the occurrence or severity of one or more symptoms of a tumor necrosis factor- α (TNF- α) -related inflammatory condition.

30. Use of a mixture according to claim 29, wherein the polynucleotide delivery-enhancing polypeptide 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: 1MW20,000-50,000, poly (Orn, Trp) 4: 120,000-50,000, melittin, histone H1, histone H3 and histone H4, SEQ ID NOS 27-31, 35-42, 45, 47, 50-59, 62, 63, 67, 68, 73, 74, 76, 78-87, 89-92, 94-108, 164-178 and 180-186.

31. The use of a mixture according to claim 29, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is acetyl.

32. The use of a mixture according to claim 29, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide 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 a tumor necrosis factor-alpha (TNF- α) gene.

34. The use of the mixture of claim 29 wherein the dsRNA is an siRNA consisting of about 10 to about 40 base pair sequence selected from the group consisting of SEQ ID NOS 109-163 and 187.

35. The use of the mixture of claim 29, wherein the polynucleotide delivery-enhancing polypeptide is admixed, combined or conjugated with the dsRNA.

36. The use of a mixture of claim 29, wherein the polynucleotide delivery-enhancing polypeptide binds to the dsRNA.

37. The use of the mixture of claim 29, wherein the animal subject is a mammal.