JP2009507852A - Pharmaceutical composition for delivery of ribonucleic acid to cells - Google Patents

Abstract

【Task】
[Solution]
Compositions, methods for producing uptake of double stranded RNA (dsRNA) and reduction of target mRNA in animal cells, and of tumor necrosis factor-alpha (TNF-α) related inflammatory conditions in animal subjects The use of a mixture comprising an amphiphilic and nucleic acid binding polynucleotide delivery-enhancing polypeptide and dsRNA in the manufacture of a therapeutic medicament is described.
[Selection] Figure 5

Description

  The present invention relates to methods and compositions for delivering nucleic acids into cells. More specifically, the present invention relates to methods and formulations for delivering double-stranded polynucleotides into cells to alter the expression of target genes and alter phenotypes such as cellular pathology or potential. .

Background of the Invention Delivery of nucleic acids to animal and plant cells has long been an important issue in molecular biology research and development. With recent developments in the areas of gene therapy, antisense therapy and RNA interference (RNAi) therapy, there is a need for the development of more effective methods for introducing nucleic acids into cells.

  A wide variety of plasmids and other nucleic acid “vectors” have been developed to deliver large polynucleotide molecules into cells. Typically, these vectors incorporate large DNA molecules comprising intact genes for transforming target cells to express a gene for scientific or therapeutic purposes.

  The process by which exogenous nucleic acid is artificially delivered into cells is commonly referred to as transfection. A variety of techniques and materials can be used to transfect cells to incorporate functional nucleic acid from an exogenous source. The most widely used transfection methods are the calcium phosphate transfection method and the electroporation method. Various other methods for transducing cells to deliver exogenous DNA or RNA molecules have also been developed, including virus-mediated transduction methods, cationic lipid delivery methods or liposome delivery methods, and mechanical or Numerous methods aimed at biochemical membrane disruption / permeation (eg, by using a surfactant, microinjection, or particle gun) are included.

  RNA interference is a sequence-specific post-transcriptional gene silencing in cells initiated by a double-stranded (ds) polynucleotide, usually dsRNA, that is homologous to a portion of the target messenger RNA (mRNA). Is a process. Introduction of the appropriate dsRNA into the cell results in the destruction of endogenous, homologous mRNA (ie, mRNA that shares substantial sequence identity with the introduced dsRNA). The dsRNA molecule is cleaved by a RNase III family nuclease called Dicer into short interfering RNA (siRNA) having a length of 19 to 23 nucleotides (nt). The siRNA is then incorporated into a multi-component nuclease complex known as an RNA-induced silencing complex or “RISC”. The RISC achieves silencing of gene expression by identifying mRNA substrates through their homology to siRNA and binding to and destroying the target mRNA.

  RNA interference has emerged as a promising technique for altering the expression of specific genes in plant and animal cells, thus treating a wide range of diseases and disorders that can be treated by modifying the expression of foreign genes It is expected to provide useful tools for doing so.

  In particular, in view of the fact that existing techniques for delivering nucleic acids to cells are limited by the poor efficiency and / or antitoxicity of delivery reagents, siRNA and other small inhibitory nucleic acids (small inhibitory nucleic acids) are introduced into cells. There remains a long-standing need in the art for better tools and methods for delivering nucleic acid (siNA). It also uses non-toxic delivery vehicles to deliver effective amounts of siNA to selected cells, tissues, or compartments in an active and durable state to alter the target cell phenotype or pathology. There is also a related need for improved methods and formulations to mediate the regulation of gene expression.

SUMMARY OF THE INVENTION One aspect of the present 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 has nucleic acid binding properties. With In a related embodiment, the polynucleotide delivery-enhancing polypeptide comprises about 5 to about 40 amino acids and has a molecular weight of 20,000 to 50,000 poly (Lys, Tryp) 4: 1, 20,000 to 50 , Poly (Orn, Trp) 4: 1, melittin, histone H1, histone H3 and histone H4, SEQ ID NOs: 27 to 31, 35 to 42, 45, 47, 50 to 59, 62, 63, 67, 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178, and 180 to 186.

  In another embodiment, the composition causes uptake of dsRNA into animal cells. 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 yet another embodiment, the dsRNA is a small interfering ribonucleic acid comprising 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. ) (SiRNA). In a related embodiment, the dsRNA is a siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID NOs: 109 to 163 and 187. In another embodiment, the polynucleotide delivery-enhancing polypeptide is mixed with, complexed with or bound to the dsRNA. In another embodiment, the polynucleotide delivery-enhancing polypeptide binds to the dsRNA. In yet another embodiment, any of the above compositions further comprises a cationic lipid. In a related embodiment, the cationic lipid is N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride, 1,2-bis (oleoyloxy). -3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2 (sperminecarboxamide) Ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid, 1,3-dioleoyloxy-2- (6 carboxyspermyl) propylamide, 5-carboxyspermylglycine dioctadecylamide, tetramethyl Tetrapalmitoylspermine, tetramethyltetrao Ilspermine, tetramethyltetralaurylspermine, tetramethyltetramyristylspermine and tetramethyldioleylspermine, DOTMA (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride ), DOTAP (1,2-bis (oleoyloxy) -3,3- (trimethylammonium) propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide), DDAB (dimethyldimethyl). Octadecylammonium bromide), polyvalent cationic lipid, lipospermine, DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid) DOSPER (1,3-dioleoyloxy-2- (6 carboxyspermyl) propylamide) (l, 3-dioleoyloxy-2- (6 carboxylic spermyl) -propyl-amid), di- and tetra-alkyl-tetra- Methyl spermine, TMPPS (tetramethyltetrapalmitoyl spermine), TMTSOS (tetramethyltetraoleyl spermine), TMTLS (tetramethyltetralauryl spermine), TTMMS (tetramethyltetramyristyl spermine), TMDOS (tetramethyldioleyl spermine) DOGS (dimethyl) Octadecylamidoglycylspermine (TRANSFECTAM®), cationic lipid combined with non-cationic lipid, DOPE Cationic lipid composition consisting of a 3: 1 (w / w) mixture of oleoyl phosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol, DOSPA and DOPE, and 1: 1 (w / W) selected from the group consisting of mixtures.

  Another aspect of the invention results in the incorporation of double-stranded ribonucleic acid (dsRNA) into animal cells, the method comprising incubating the animal cells with a mixture comprising the polynucleotide delivery polypeptide and dsRNA. Prepare. The polynucleotide delivery polypeptide is amphiphilic and has nucleic acid binding properties.

  Another aspect of the present invention is a method of altering the expression of a target gene in an animal cell that is amphipathic and nucleic acid binding, the method comprising a polynucleotide delivery-enhancing polypeptide comprising: The polynucleotide delivery polypeptide is amphipathic and has a nucleic acid binding property, and a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA is complementary to a region of the target gene. Incubating the animal cells with a mixture comprising a double-stranded ribonucleic acid (dsRNA).

  A related embodiment is any of the above methods, wherein the animal cell is a mammalian cell.

  Another aspect of the invention is a method of altering the phenotype of a subject animal, wherein the method is a polynucleotide delivery-enhancing polypeptide, wherein the polynucleotide delivery-enhancing polypeptide is amphiphilic and nucleic acid binding A polynucleotide delivery-enhancing polypeptide having sex and a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA is complementary to a region of a target gene in a subject. Administering the mixture to the subject animal. In a related embodiment, the animal may be a mammal.

  Another embodiment is the method of any of the above, wherein the polynucleotide delivery-enhancing polypeptide comprises about 5 to about 40 amino acids and has a molecular weight of 20,000 to 50,000 (Lys, Tryp) 4: 1, 20,000 to 50,000 poly (Orn, Trp) 4: 1, melittin, histone H1, histone H3 and histone H4, SEQ ID NOs: 27 to 31, 35 to 42, 45, 47, 50 to 59 62, 63, 67, 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178, and 180 to 186. A related embodiment is any of the above methods, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is acetylated. Another related embodiment is any of the methods described above, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is PEGylated. Another embodiment is any of the above methods, wherein the dsRNA is a small molecule 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. It is a small interfering ribonucleic acid (siRNA). A related embodiment is any of the above methods, wherein the dsRNA is a siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID NOs: 109 to 163 and 187. Another embodiment is any of the methods described above, wherein the polynucleotide delivery-enhancing polypeptide is mixed with, complexed with, or bound to the dsRNA. Yet another embodiment is any of the methods described above, wherein the polynucleotide delivery-enhancing polypeptide binds to the dsRNA. In another embodiment, the method of any of the above, further comprising a cationic lipid. A related embodiment is any of the above methods, wherein the cationic lipid is N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride, 1 , 2-bis (oleoyloxy) -3-3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy -N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid, 1,3-dioleoyloxy-2- (6 carboxyspermyl) propylamide, 5-carboxy Spermylglycine dioctadecylamide, tetramethyltetrapalmitoyl sp , Tetramethyltetraoleylspermine, tetramethyltetralaurylspermine, tetramethyltetramyristylspermine and tetramethyldioleylspermine, DOTMA (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride), DOTAP (1,2-bis (oleoyloxy) -3,3- (trimethylammonium) propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide) , DDAB (dimethyldioctadecylammonium bromide), polyvalent cationic lipid, lipospermine, DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propana Minium trifluoroacetic acid), DOSPER (1,3-dioleoyloxy-2- (6 carboxyspermyl) propylamide) (l, 3-dioleoyloxy-2- (6 carboxylic spermyl) -propyl-amide), di-and Tetra-alkyl-tetra-methylspermine, TMPPS (tetramethyltetrapalmitoylspermine), TMTOS (tetramethyltetraoleylspermine), TMTLS (tetramethyltetralaurylspermine), TTMMS (tetramethyltetramyristylspermine), TMDOS (tetramethyldio) Railspermine) DOGS (dioctadecylamidoglycylspermine (TRANSFECTAM®), conjugated with non-cationic lipids Cationic lipid compositions consisting of a 3: 1 (w / w) mixture of thionic lipids, DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol, DOSPA and DOPE, and DOTMA and Selected from the group consisting of a 1: 1 (w / w) mixture of DOPE.

  Another aspect of the invention is a polynucleotide delivery polypeptide, wherein the polynucleotide delivery polypeptide is amphipathic and has nucleic acid binding properties, and tumor necrosis in the subject animal. A drug for the treatment of factor-alpha (TNF-α) -related inflammatory disorders, which reduces TNF-α RNA levels, thereby preventing the onset or severity of one or more symptoms of TNF-alpha-related inflammatory disorders Use of a mixture comprising double-stranded ribonucleic acid (dsRNA) for the manufacture of a drug that can be or can be mitigated. In one embodiment, the polynucleotide delivery-enhancing polypeptide comprises about 5 to about 40 amino acids and has a molecular weight of 20,000 to 50,000 poly (Lys, Tryp) 4: 1, 20,000 to 50,000. 000 poly (Orn, Trp) 4: 1, melittin, histone H1, histone H3 and histone H4, SEQ ID NOS: 27 to 31, 35 to 42, 45, 47, 50 to 59, 62, 63, 67, 68, 73 , 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178 and 180 to 186. In a related embodiment, the N-terminus of the 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 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. (SiRNA). In a related embodiment, the dsRNA is a siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID NOs: 109 to 163 and 187. In another embodiment, the polynucleotide delivery-enhancing polypeptide is mixed, complexed or bound with the dsRNA. In another embodiment, the polynucleotide delivery-enhancing polypeptide binds to the dsRNA. In another embodiment, the target animal is a mammal.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION The present invention provides novel compositions and methods that use short interfering nucleic acids (siNA), or precursors thereof, in combination with polynucleotide delivery-enhancing polypeptides. By providing, the above-mentioned demand is satisfied and further problems and advantages are satisfied. A polynucleotide delivery-enhancing polypeptide is a natural or artificial polypeptide selected for its ability to promote intracellular delivery or uptake of a polynucleotide comprising siNA and its precursors.

  In the novel compositions of the present invention, siNA is mixed, complexed or conjugated with a polynucleotide delivery-enhancing polypeptide so that the target cell becomes naked siNA (ie, the delivery-enhancing polypeptide is A composition that facilitates intracellular delivery of siNA can be formed as compared to delivery resulting from contact with non-existing siNA).

  In certain embodiments of the invention, the polynucleotide delivery-enhancing polypeptide is a histone protein, or a polypeptide or peptide fragment, derivative, analog, or conjugate thereof. In these embodiments, the siNA is one or more full-length histone proteins, or a polypeptide corresponding at least in part to a partial sequence of histone proteins, such as one or more of the following histones: Mixed, complexed or bound: histone H1, histone H2A, histone H2B, histone H3 or histone H4, or typically at least 5-10 or 10 to 10 of the natural histone proteins A polypeptide fragment or derivative of one or more histones comprising at least a partial sequence of a histone protein that is 20 consecutive residues. In a more detailed embodiment, the siRNA / histone mixture, complex or conjugate is substantially free of amphiphilic compounds. In other detailed embodiments, the siNA mixed, complexed or bound with a histone protein or polypeptide has, for example, 30 nucleotides or less, and a small interfering RNA (siRNA). A double-stranded RNA such as a double-stranded RNA. In an exemplary embodiment, the histone polynucleotide delivery-enhancing polypeptide comprises a fragment of histone H2B, as exemplified by the polynucleotide delivery-enhancing polypeptide named PN73 described below. In further detailed embodiments, the polynucleotide delivery-enhancing polypeptide may be PEGylated, particularly to improve stability and / or efficiency in the context of in vivo administration.

  In a further embodiment of the invention, the polynucleotide delivery-enhancing polypeptide is selected or rationally designed to comprise an amphiphilic amino acid sequence. For example, a plurality of non-polar or hydrophobic amino acids that form a domain or motif in a hydrophobic sequence linked to a plurality of charged amino acid residues that form a domain or motif in a charged sequence to yield an amphiphilic peptide Useful polynucleotide delivery-enhancing polypeptides comprising residues can be selected.

  In other embodiments, the polynucleotide delivery-enhancing polypeptide is selected to comprise a protein transduction domain or motif, and a fusion peptide domain or motif. The protein transduction domain is a peptide sequence that can be inserted into the cell membrane, and preferably can pass through the cell membrane. A fusion peptide is a peptide that destabilizes a lipid membrane, such as a membrane that wraps a cell membrane or endosome, and this destabilization can be promoted at low pH. Typical fusion domains or motifs are found in a variety of viral fusion proteins and other proteins, such as fibroblast growth factor 4 (FGF4).

  In order to rationally design the polynucleotide delivery-promoting polypeptide of the present invention, a protein transduction domain is used as a motif that facilitates the entry of a nucleic acid into a cell through a cell membrane. In certain embodiments, the transported nucleic acid is encapsulated within an endosome. The internal pH of the endosome is low, so that the fusion peptide motif destabilizes the endosomal membrane. Due to destabilization and disruption of the endosomal membrane, siNA binds to the RISC complex and is released into the cytoplasm where it can be directed to its target mRNA.

Examples of protein transduction domains for selective incorporation into the polynucleotide delivery-enhancing polypeptides of the invention include the following:
1. TAT protein transduction domain (PTD) (SEQ ID NO: 1) KRRQRRR;
2. Penetratin PTD (SEQ ID NO: 2) RQIKIWFQNRRMKWKK;
3. VP22 PTD (SEQ ID NO: 3) DAATATRGRSASARPRPRAPPARSASRPRPVD;
4). Kaposi FGF signal sequence (SEQ ID NO: 4) AAVALLPAVLLALLAP and SEQ ID NO: 5) AAVLLPVLLPVLLAAP;
5. Human integrin β3 signal sequence (SEQ ID NO: 6) VTVLALGALAGVGVG;
6). a gp41 fusion sequence (SEQ ID NO: 7) GALFLGWWLGAAGSTMGA;
7). Caiman crocodylus Ig (v) light chain (SEQ ID NO: 8) MGLGLHLLVLAAALQGA;
8). hCT-derived peptide (SEQ ID NO: 9) LGTYTQDFNKFHTFPQTAIGVGAP;
9. Transportan (SEQ ID NO: 10) GWTLNSAGYLLKINLKALAALAKIL;
10. Loligomer (SEQ ID NO: 11) TPPKKRKVEDPKKKK;
11. 11. Arginine peptide (SEQ ID NO: 12) RRRRRRR; Amphipathic model peptide (SEQ ID NO: 13) KLALKLALKALKAALKLA.

Examples of viral fusion peptide fusion domains for selective incorporation into the polynucleotide delivery-enhancing polypeptides of the invention include the following:
1. Influenza HA2 (SEQ ID NO: 14) GLFGAIAGFIENGWEEG;
2. Sendai F1 (SEQ ID NO: 15) FFGAVIGITALGVATA;
3. Respiratory syncytial virus F1 (SEQ ID NO: 16) FLGFLLLGVGSAIASGV;
4). 4. HTV gp41 (SEQ ID NO: 17) GVFVLGFLGLATAGGS; Ebola GP2 (SEQ ID NO: 18) GAAIGLAWIPYFGPAA.

  In a further embodiment of the invention, a poly-incorporated DNA binding domain or motif that promotes polypeptide-siNA complex formation and / or improves delivery of siNA within the methods and compositions of the invention. Nucleotide delivery enhancing polypeptides are provided. Exemplary DNA binding domains in this sense include various “zinc finger” domains, as described for the DNA binding regulatory proteins and other proteins identified in Table 1 below. (See, eg, Simpson et al., J. Biol. Chem, 278: 28011-28018, 2003).

Table 1:

* The table above binds to double-stranded DNA characterized by the motif pattern of Cx (2,4) -Cx (12) -Hx (3) -H (SEQ ID NO: 188) Conservative zinc fingers are shown for use with which it is possible to select and design additional polynucleotide delivery-enhancing polypeptides according to the invention.
** The sequences shown in Table 1, Sp1, Sp2, Sp3, Sp4, DrosBtd, DrosSp, CeT22C8.5, and Y4pB1A. 4 is defined in the present specification as SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, and 26, respectively.

  Alternative DNA binding domains useful for constructing the polynucleotide delivery-enhancing polypeptides of the invention include, for example, a portion of the HIV Tat protein sequence (see examples below).

  In an exemplary embodiment of the invention described herein below, the polynucleotide delivery-enhancing polypeptide is effective in improving siNA delivery to target cells with any of the aforementioned structural elements, domains or motifs. Can be rationally designed and constructed by combining them into a single polypeptide that mediates. For example, by fusing the protein transduction domain of a TAT polypeptide to the N-terminal 20 amino acids of a hemagglutinin protein called HA2 of influenza virus, one representative polynucleotide delivery-enhancing polypeptide herein is Obtained. The present disclosure presents a variety of other polynucleotide delivery-enhancing polypeptide constructs that can be widely applied to create a wide variety of polynucleotide delivery-enhancing polypeptides that are effective in enhancing siNA delivery. It is clearly stated that it can be used.

Further representative polynucleotide delivery-enhancing polypeptides that are within the scope of the present invention can be selected from the following peptides:
WWETWKPFQCRICMRNFSTRQARRNNHRRRRHR (SEQ ID NO: 27);
GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO: 29), GRKKRRQRRRRPPQGRKKRRQRRRRPPQGRKKRRQRRRRPPQ (SEQ ID NO: 30), GEQIAQLIAG000ID30K, GEQIAQLIAGYIDK 000 to 50,000 poly Orn-Trp, 4: 1. Additional useful polynucleotide delivery-enhancing polypeptides within the compositions and methods herein include all or part of the melittin protein sequence.

  Yet another representative polynucleotide delivery-enhancing polypeptide is identified in the Examples below. Selecting or combining any one or combination of these peptides effectively induces or enhances delivery of siNA in the cell within the methods and compositions of the invention. Polypeptide reagents can be obtained.

  In a more detailed aspect of the invention, the mixture, complex or conjugate comprising siRNA and polynucleotide delivery-enhancing polypeptide is optionally combined with a cationic lipid, such as LIPOFECTIN®. (For example, mixed or complexed). In this sense, it is unexpectedly provided that a polynucleotide delivery-enhancing polypeptide complexed or bound to siRNA alone achieves delivery of siNA sufficient to mediate gene silencing by RNAi. It is disclosed that it will do. However, it is further unexpected herein that siNA delivery when siRNA / polynucleotide delivery-enhancing polypeptide complexes or conjugates are mixed or complexed with cationic lipids such as lipofectin. And it has also been disclosed that it will exhibit a more potent activity in mediating gene silencing. To create these compositions comprised of polynucleotide delivery-enhancing polypeptides, siRNA and cationic lipids, the siRNA and peptide may first be mixed together in a suitable medium such as a cell culture medium. The cationic lipid is then added to the mixture to form a siRNA / delivery peptide / cationic lipid composition. Optionally, the peptide and cationic lipid are first mixed together in a suitable medium, such as a cell culture medium, and then the siRNA is added to form a siRNA / delivery peptide / cationic lipid composition. May be.

  Examples of useful cationic lipids within the scope of embodiments of the present invention include: N- [1- (2,3-dioleoyloxy) propyl] -N, N, N -Trimethylammonium chloride, 1,2-bis (oleoyloxy) -3-3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide, 2,3-dioleoyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid, 1,3-dioleoyloxy-2- (6 carboxyspermyl) ) Propylamide, 5-carboxyspermylglycine dioctadecylamide, tetramethyltetrapa Mitoylspermine, tetramethyltetraoleylspermine, tetramethyltetralaurylspermine, tetramethyltetramyristylspermine and tetramethyldioleylspermine, DOTMA (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride), DOTAP (1,2-bis (oleoyloxy) -3,3- (trimethylammonium) propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium) Bromide) or DDAB (dimethyldioctadecyl ammonium bromide). Polyvalent cationic lipids include lipospermine, specifically, DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propana Minium trifluoroacetic acid) and DOSPER (1,3-dioleoyloxy-2- (6 carboxyspermyl) propylamide) (l, 3-dioleoyloxy-2- (6 carboxylic spermyl) -propyl-amide), and di- And tetra-alkyl-tetra-methyl spermine, TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethyltetralauryl spermine), TTMMS (tetramethyltetramyristyl spellin) ) And TMDOS (tetramethyldioleylspermine), DOGS (dioctadecylamidoglycylspermine (TRANSFECTAM®)), but not limited thereto. Cationic lipids include, for example, US Pat. No. 6,733,777; US Pat. No. 6,376,248; US Pat. No. 5,736,392; US Pat. No. 5,686,958; No. 5,334,761; and US Pat. No. 5,459,127.

  Cationic lipids may be selectively combined with non-cationic lipids, particularly neutral lipids such as lipids such as DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanyl phosphatidylethanolamine) or cholesterol. . Cationic lipid composition consisting of a 3: 1 (w / w) mixture of DOSPA and DOPE, or a 1: 1 (w / w) mixture of DOTMA and DOPE (LIPOFECTIN®, Invitrogen) The cationic lipid compositions that are made are generally useful for transfecting the compositions of the invention. Preferred transfection compositions are those that promote substantial transfection in higher eukaryotic cell lines.

  In an exemplary embodiment, the invention provides a short interfering nucleic acid (siNA), mixed or conjugated or conjugated with a polynucleotide delivery-enhancing polypeptide. Features a composition comprising a low molecular weight nucleic acid molecule, such as short interfering RNA (siRNA), double stranded RNA (dsRNA), microRNA (mRNA), or short hairpin RNA (shRNA) And

  As used herein, “Short Interfering Nucleic Acid”, “siNA”, “Short Interfering RNA”, “siRNA”, “Short Interfering Nucleic Acid Molecules” acid molecule) "," short interfering oligonucleotide molecule ", or" chemically-modified short interfering nucleic acid molecule, e.g., the term "specifically," specifically referred to as a chemically modified short interfering nucleic acid molecule, Mediates RNA interference "RNAi" or gene silencing By any nucleic acid molecule capable of inhibiting or down-regulating gene expression or viral replication. In an exemplary embodiment, the siNA is a self-complementary sense region and antisense region, wherein the antisense region is a nucleotide sequence within a nucleic acid molecule that is targeted for down-regulation of expression or Including a nucleic acid sequence complementary to a portion thereof, the sense region comprising a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (ie, substantially identical in sequence to the target nucleic acid molecule) A double-stranded polynucleotide molecule comprising such a region.

  “SiNA” is a short-length double-stranded nucleic acid (which may be a longer precursor thereof) and does not exhibit unacceptable toxicity in target cells, for example It means a small interfering nucleic acid such as siRNA. Useful siNA lengths within the scope of the present invention will be optimized in certain embodiments with a length of about 21 to 23 bp. However, there is no specific limitation on the length of useful siNA comprising siRNA. For example, siNA is initially in a final form that exhibits gene silencing activity upon delivery into or after delivery into target cells, or a precursor form that is significantly different from the processed form of siNA. Can be provided to cells. The precursor form of siNA is processed, degraded, altered, or cleaved during or after delivery, for example, to generate siNA that exhibits activity in mediating gene silencing in the cell. Precursor array elements may be included. Thus, in certain embodiments, useful siNAs within the scope of the present invention are, for example, about 100-200 base pairs, 50-100 base pairs, or 50-100 base pairs, which produce active processed siNAs in the target cell, or It will have a precursor length of less than about 50 base pairs. In other embodiments, useful siNA or siNA precursors will be about 10 to 49 bp, 15 to 35 bp, or about 21 to 30 bp in length.

  In certain embodiments of the invention, as noted above, the use of polynucleotide delivery-enhancing polypeptides facilitates delivery of larger nucleic acid molecules than conventional siNA with siNA giant nucleic acid precursors. . For example, the methods and compositions herein can be used to facilitate delivery of a large nucleic acid representative of a “precursor” to a desired siNA, where the precursor amino acid is directed to a target cell. Can be cleaved before, during or after delivery, or otherwise processed to form active siNA to modulate gene expression in target cells. For example, a siNA precursor polypeptide has two or more loop structures and self-complementary sense and antisense regions, wherein the antisense region is responsible for down-regulation of expression. A stem comprising a region comprising a nucleic acid sequence complementary to a nucleotide sequence or a part thereof in a target nucleic acid molecule, wherein the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof A circular single-stranded polynucleotide having a (stem), wherein the circular polynucleotide is processed in vivo or in vitro to yield an active siNA molecule capable of mediating RNAi. It can be selected as a nucleotide.

  In mammalian cells, dsRNAs exceeding 30 base pairs are usually associated with interferon-induced dsRNA-dependent kinase PKR (dsRNA-dependent kinase PKR) and 2′-5′-oligoadenylate synthetase (2 ′ -5'-oligoadenylate synthetase) can be activated. Activated PKR inhibits overall translation by phosphorylation of the translation factor eukaryotic initiation factor 2α (eIF2α), while 2′-5′-oligoadenylate. Synthetase causes non-specific mRNA degradation through activation of RNase L. Due to its small size, usually less than 30 base pairs and most commonly between about 17-19, 19-21, or 21-23 base pairs (especially referring to non-precursor forms) The siNA of the present invention avoids activation of the interferon response.

  In contrast to the non-specific effects of long dsRNA, siRNA can mediate selective gene silencing in mammalian systems. Hairpin RNAs with short loops and 19-27 base pairs in the stem can also selectively silence the expression of genes homologous to sequences in the double stranded stem. Mammalian cells can convert short hairpin RNA to siRNA to mediate selective gene silencing.

  RISC mediates cleavage of single stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs at the center of the region complementary to the antisense strand of the siRNA duplex. Studies have shown that 21 nucleotide siRNA duplexes are most active when equipped with a 2 nucleotide 3 'overhang. Furthermore, complete replacement of one or both siRNA strands with 2'-deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi activity, while deoxynucleotides (2'-H) Substitution of overhanging nucleotides on the 3 ′ end of siRNA by has been reported to be permissible.

  Studies have shown that replacement of the 3 'overhang segment of a 21-mer siRNA duplex with a 2 nucleotide 3' overhang by deoxyribonucleotides does not adversely affect RNAi activity. Substitution of 4 nucleotides or less at each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, but complete substitution with deoxyribonucleotides results in loss of RNAi activity.

  Alternatively, siNA can be delivered as single or multiple transcripts expressed by polynucleotide vectors that encode single or multiple siNAs and express these siNAs in target cells. . In these embodiments, the double stranded portion of the final transcript of siRNA expressed in the target cell will be, for example, 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bp in length. In an exemplary embodiment, the duplex portion where the two strands of siNA are paired is not limited to a completely paired nucleotide segment, but a mismatch (the corresponding nucleotide is complementary) Not), bulges (lack of complementary nucleotides corresponding to one strand), overhangs, etc., may be included. Non-paired portions may be included to the extent that they do not interfere with siNA formation. In a more detailed embodiment, the “bulge” may contain 1 to 2 unpaired nucleotides, and the double stranded region in which the duplexes of siRNA paired comprises about 1 to 7 There may be one or about 1 to 5 bulges. In addition, there may be a total of about 1 to 7 or about 1 to 5 “mismatch” portions contained within the siNA duplex region. The most frequent mismatch is when one of the nucleotides is guanine and the other is uracil. Such a mismatch is caused by, for example, a mutation from C to T, G to A, or a mixture thereof in the corresponding DNA encoding the sense RNA, but other factors are also conceivable. Furthermore, in the present invention, the double-stranded region in which two strands of siNA are paired includes both a bulge portion and a mismatched portion in a range close to the specified numerical range. Also good.

  The terminal structure of the siNA of the present invention may be either smooth or adherent (having an overhang) as long as the siNA retains its activity and silences the expression of the target gene. Adhesive (with overhangs) end structures are not limited to 3 'overhangs as reported by others. On the contrary, a 5 'overhang structure may be included as long as gene silencing action by RNAi or the like can be induced. In addition, the number of overhanging nucleotides is not limited to the limit of 2 or 3 nucleotides as reported, as long as the overhang does not impair the gene silencing activity of siNA. It may be. For example, the overhang may comprise about 1 to 8 nucleotides, more often about 2 to 4 nucleotides. The total length of siNA having a cohesive end structure is expressed as the sum of the length of the paired double-stranded portion and the length of the pair comprising a single strand overhanging at both ends. For example, in a typical example of a 19 bp double-stranded RNA having 4 nucleotide overhangs at both ends, the total length is expressed as 23 bp. Furthermore, since the overhanging sequence may exhibit low specificity for the target gene, the sequence need not be complementary (antisense) or identical (sense) to the target gene sequence. . Furthermore, as long as siNA can maintain the gene silencing action on its target gene, the siNA has a low molecular weight structure (for example, a natural RNA such as tRNA, rRNA or viral RNA, for example, in an overhang portion at one end). Molecule or artificial RNA molecule).

  Furthermore, the terminal structure of siNA may have a stem-loop structure in which one end of a double-stranded nucleic acid is linked by a linker nucleic acid such as a linker RNA. The length of the double stranded region (stem-loop portion) may be, for example, 15 to 49 bp, often 15 to 35 bp, and more generally about 21 to 30 bp in length. Alternatively, the length of the double-stranded region that is the final transcript of siNA expressed in the target cell is, for example, about 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bp. May be. When a linker segment is used, the length of the linker is not particularly limited as long as the linker does not interfere with stem pairing. For example, the linker part may have a cloverleaf tRNA structure for stable pairing of the stem part and suppression of recombination between DNAs encoding the stem part. Even if the linker has a length that prevents stem pairing, for example, it is possible to construct the linker moiety to include an intron, so that the intron is matured from the precursor RNA. It is excised during processing towards RNA, which allows stem pairing. In the case of stem-loop siRNA, any end (head or tail) that does not have the RNA loop structure may have low molecular weight RNA. As described above, these low molecular weight RNAs may include natural RNA molecules such as tRNA, rRNA or viral RNA, or artificial RNA molecules.

  The siNA may also comprise a single stranded polynucleotide having a nucleotide sequence that is complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof (eg, such siNA molecule is contained within the target nucleic acid within the siNA molecule). The single-stranded polynucleotide is a 5 ′ phosphate (see, eg, Martinez et al., Cell. 110: 563-574, 2002, where the presence of a nucleotide sequence corresponding to the sequence or a portion thereof is not required). And Schwarz et al., Molecular Cell 10: 537-568, 2002) or may further comprise a terminal phosphate group, such as 5 ′, 3′-2 phosphate.

  As used herein, the term siNA molecule is not limited to molecules comprising only natural RNA or DNA, but also encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, small interfering nucleic acid molecules of the invention lack 2'-hydroxy (2'-OH) containing nucleotides. In certain embodiments, small interfering nucleic acids do not require the presence of a nucleotide having a 2′-hydroxy group to mediate RNAi, and thus the small interfering nucleic acid molecules of the present invention are themselves ribosomal. There may be no nucleotides (eg, nucleotides having a 2′-OH group). However, siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to assist RNAi have a linked linker or linker, or other linked or associated groups, moieties, or 2′-OH 1 Alternatively, it may have a chain comprising two or more nucleotides. The siNA molecule may also optionally include ribonucleotides at 5, 10, 20, 30, 40, or 50% nucleotide positions.

  As used herein, the term siNA refers to, for example, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (mRNA), short hairpin ( short hairpin RNA) (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified RNA, short interfering nucleic acid, short interfering nucleic acid Silencing RNA (ptgsRNA), and Other such, are intended to be equivalent to other terms used to describe nucleic acid molecules capable of mediating sequence specific RNAi.

  In other embodiments, siNA molecules for use within the scope of the present invention may comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are nucleotides or non-senses. Covalently linked by a nucleotide linker molecule or alternatively non-covalently linked by ionic, hydrogen bonding, van der Waals, hydrophobic, and / or stacking interactions Yes.

  “Antisense RNA” is an RNA strand that has a sequence complementary to mRNA of a target gene and is considered to induce RNAi by binding to the mRNA of the target gene. “Sense RNA” has a sequence complementary to the antisense RNA, and anneals to the complementary antisense RNA to form siRNA. These antisense RNAs and sense RNAs have been conventionally synthesized using an RNA synthesizer.

  As used herein, the term “RNAi construct” is a general term used throughout the specification and includes small interfering RNAs (siRNAs), hairpin RNAs, and other that can be cleaved in vivo to form siRNAs. Contains RNA species. As used herein, an RNAi construct refers to an expression vector (also referred to as an RNAi expression vector) capable of producing a transcript that forms dsRNA or hairpin RNA in a cell and / or a transcript that can produce siRNA in vivo. Also has. The siRNA may optionally comprise a single strand or double strand of the siRNA.

  An si hybrid molecule is a double-stranded nucleic acid having a function similar to that of siRNA. Instead of double-stranded RNA molecules, si hybrids are composed of one RNA strand and one DNA strand. Since the antisense strand is the strand that binds to the target mRNA, preferably the RNA strand is the antisense strand. Si hybrids created by hybridization of DNA and RNA strands have hybridized complementary portions and preferably at least one 3 'overhang end.

  The siNA used within the scope of the present invention can be assembled from two separate oligonucleotides, one strand being the sense strand and the other strand being the antisense strand, in which case the antisense strand and the sense strand The strands are self-complementary (ie, each strand contains a nucleotide sequence that is complementary to the nucleotide sequence in the other strand; the antisense and sense strands are double or double) Forming a chain structure, for example, in this case, the double-stranded region is about 19 base pairs). The antisense strand may include a nucleotide sequence that is complementary to a nucleotide sequence within the target nucleic acid molecule or portion thereof, and the sense strand may include a nucleotide sequence that corresponds to the target nucleic acid sequence or portion thereof. . Alternatively, siNA can also be assembled from a single oligonucleotide, in which case the siNA's self-complementary sense and antisense regions are nucleic acid-based or non-nucleic acid-based linkers. It is connected by.

  In a further embodiment, the siNA for intracellular delivery according to the methods and compositions of the invention comprises a duplex, asymmetric duplex, hairpin, or asymmetric hairpin second having a self-complementary sense region and an antisense region. A polynucleotide having the following structure, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence within an individual target nucleic acid molecule or portion thereof, and the sense region comprises the target It comprises a nucleotide sequence corresponding to a nucleic acid sequence or a part thereof.

  Non-limiting examples of chemical modifications that can be made within siNA include phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methylribonucleotides, 2'-deoxy-2'-fluororibonucleotides. , “Universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and incorporation of terminal glyceryl and / or inverted deoxyabasic residues. It is not something. These chemical modifications have been shown to dramatically improve the stability of these compounds in serum while protecting RNAi activity in cells when used in various siNA constructs.

  In a non-limiting example, the introduction of chemically modified nucleotides into a nucleic acid molecule provides a powerful tool that overcomes the potential limitations of in vivo stability and bioavailability inherent in natural RNA molecules delivered from outside the body. provide. Because chemically modified nucleic acid molecules tend to have longer half-lives in serum, for example, using chemically modified nucleic acid molecules can be compared with a specific nucleic acid molecule for any therapeutic effect Small doses are possible. Still further, certain chemical modifications can improve the bioavailability of nucleic acid molecules by specific cells or tissues of the target and / or improve uptake of the nucleic acid molecules by cells. Thus, even if the activity of a chemically modified nucleic acid molecule is reduced compared to a natural nucleic acid molecule, eg, as compared to all RNA nucleic acid molecules, the modified nucleic acid The activity of the whole molecule may exceed the activity of the natural molecule due to its improved stability and / or delivery of the molecule. Unlike natural unmodified siNA, chemically modified siNA can also minimize the possibility of activating interferon activity in the human body.

  In the siNA molecules described herein, the antisense region of the siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3 'end of the antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about 1 to about 5 phosphorothioate internucleotide linkages at the 5 'end of the antisense region. In any of the embodiments of siNA molecules described herein, the 3 ′ terminal nucleotide overhang of the siNA molecule of the invention comprises a ribonucleotide or deoxyribonucleotide in which the sugar, base, or backbone of the nucleic acid is chemically modified. I can. In any of the embodiments of siNA molecules described herein, the 3 'terminal nucleotide overhang can comprise one or more universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3 'terminal nucleotide overhang can comprise one or more acyclic nucleotides.

  For example, in a non-limiting example, the present invention provides 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages within a single molecular interference nucleic acid (siNA) strand. Features chemically modified small interfering nucleic acids (siNA). In yet another embodiment, the present invention independently comprises 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both small interfering nucleic acid (siNA) strands. Characterized by chemically modified small interfering nucleic acids (siNA). The phosphorothioate internucleotide linkage may be present in one or both oligonucleotide strands of the siNA duplex, eg, in the sense strand, in the antisense strand, or in both strands. . The siNA molecules of the present invention have one or more phosphorothioate internucleotide linkages at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the sense strand, antisense strand, or both strands. May be included. For example, exemplary siNA molecules of the invention have from about 1 to about 5 or more (eg, about 1, 2, 3, 4, etc.) at the 5 ′ end of the sense strand, antisense strand, or both 5 or more) may comprise consecutive phosphorothioate internucleotide linkages. In another non-limiting example, a representative siNA molecule of the invention has one or more (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) may include pyrimidine phosphorothioate internucleotide linkages. In yet another non-limiting example, a representative siNA molecule of the invention has one or more (eg, about 1, 2, 3, 4) in the sense strand, antisense strand, or both strands. 5, 6, 7, 8, 9, 10 or more) may contain purine phosphorothioate internucleotide linkages.

  The siNA molecule may be composed of a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (eg, about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides. Having a base pair of about 18 to about 23 (eg, about 18, 19, 20, 21, 22, or 23), wherein the circular oligonucleotide comprises about 19 base pairs and A dumbbell structure with two loops is formed.

  A circular siNA molecule comprises two loop motifs, but one or both loop portions of the siNA molecule are biodegradable. For example, a circular siNA molecule of the present invention can be obtained with two 3 ′ terminal overhangs, such as a 3 ′ terminal nucleotide overhang containing about 2 nucleotides, due to degradation of the loop portion of the siNA molecule in vivo. Designed to be capable of generating chain siNA molecules.

  Modified nucleotides present in the siNA molecule, preferably in the antisense strand of the siNA molecule, but also selectively in the sense strand and / or in both the antisense and sense strands are similar to natural ribonucleotides Modified nucleotides having the specified properties or characteristics. For example, the present invention includes modified nucleotides having a Northern configuration (eg, the Northern pseudocycle), such as Saenger, “Principles of Nucleic Acid Cycle ) ", Edited by Springer-Verlag, 1984), characterized by siNA molecules. A chemically modified nucleotide present in the siNA molecule of the present invention, preferably in the antisense strand of the siNA molecule of the present invention, as well as selectively in the sense strand and / or in both the antisense and sense strands, As such, it is resistant to nuclease degradation while retaining the ability to mediate RNAi. Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene- (D-ribofuranosyl). ) Nucleotide); 2′-methoxyethoxy (MOE) nucleotide; 2′-methylthioethyl, 2′-deoxy-2′-fluoro nucleotide, 2′-deoxy-2′-chloronucleotide, 2′-azido nucleotide, and 2 '-O-methyl nucleotides are included.

  The sense strand of a double stranded siNA molecule has a terminal end, such as an inverted deoxybasic moiety, at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the sense strand. You may have a cap part (terminal cap moiety).

  Non-limiting examples of conjugates include Vargeese et al., Filed Apr. 30, 2003. , US patent application Ser. No. 10 / 427,160, which is incorporated herein by reference in its entirety, including drawings. In another embodiment, the conjugate is covalently linked to the chemically modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule binds to the sense strand, the antisense strand, or the 3'end of either of these strands of the chemically modified siNA molecule. In another embodiment, the conjugate molecule binds to the 5 'end of the sense strand, antisense strand, or both of these strands of a chemically modified siNA molecule. In yet another embodiment, the conjugate molecule is at both the 3 'and 5' ends of the sense strand, the antisense strand, or both of these chemically modified siNA molecules, or any combination thereof. Join. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically modified siNA molecule into a biological system such as a cell. In another embodiment, the conjugate molecule that binds to the chemically modified siNA molecule is polyethylene glycol, human serum albumin, or a ligand for a cellular receptor capable of mediating cellular uptake. Examples of specific conjugate molecules contemplated by the present invention capable of binding to chemically modified siNA molecules are described in Vargeese et al., Published July 10, 2003. , US Patent Publication No. 20030130186, and US Patent Publication No. 20040110296 published on June 10, 2004. The types of conjugates used and the range of conjugates of the siNA molecules of the present invention can improve the pharmacokinetic properties, bioavailability, and / or stability of siNA constructs while simultaneously maintaining the ability of siNA to mediate RNAi activity. It is possible to evaluate sex. One skilled in the art can, for example, screen siNA constructs modified with various conjugates in animal models generally known in the art to determine the ability of the siNA conjugate complex to mediate RNAi. It can be determined whether or not the improved properties are retained while maintaining.

  Furthermore, siNA may be composed of a nucleotide linker, a non-nucleotide linker, or a mixed nucleotide / non-nucleotide linker that links the sense region of the siNA to the antisense region of the siNA. In one embodiment, the nucleotide linker may be more than 2 nucleotides in length, eg, 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. As used herein, an “aptamer” or “nucleic acid aptamer” is a nucleic acid molecule that specifically binds to a target molecule and has a sequence comprising a sequence that is recognized by the target molecule in its natural environment. Means nucleic acid molecule. Alternatively, the aptamer may be a nucleic acid molecule that binds to the target molecule, even if the target molecule does not naturally bind to the nucleic acid. The target molecule can be any molecule of interest. For example, aptamers can be used to bind to the ligand binding domain of a protein, thereby preventing interaction with the protein by a natural ligand. However, this is a non-limiting example and those skilled in the art will recognize that other embodiments can be readily made using techniques generally known in the art. . For example, Gold et al. , Annu. Rev. Biochem. 64: 763, 1995; Brody and Gold, J .; Biotechnol. 74: 5, 2000; Sun, Curr. Opin. Mol. Ther. 2: 100, 2000; Kusser, J. et al. Biotechnol. 74:27, 2000; Hermann and Patel, Science 287: 820, 2000; and Jayasena, Clinical Chemistry 45: 1628, 1999.

  Non-nucleotide linkers can be abasic nucleotides, polyesters, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons, or other polymer compounds (eg, polyethylene glycols having 2 to 100 ethylene glycol units). ). Specific examples include Seela and Kaiser, Nucleic Acids Res. 18: 6353, 1990, and Nucleic Acids Res. 15: 3113, 1987; Cload and Schepartz, J. et al. Am. Chem. Soc. 113: 6324, 1991; Richardson and Schepartz, J. et al. Am. Chem. Soc. 113: 5109, 1991; Ma et al. , Nucleic Acids Res. 21: 2585, 1993, and Biochemistry 32: 1751, 1993; Durand et al. , Nucleic Acids Res. 18: 6353, 1990; McCurdy et al. , Nucleosides & Nucleotides 10: 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 No. WO 95/11910, and Ferentz and Verdine, J. et al. Am. Chem. Soc. 113: 4000, 1991, are included. A “non-nucleotide” is a group or compound that can be incorporated into a nucleic acid strand in place of one or more nucleotide units, comprising either sugar and / or phosphate substitutions, and the remaining base Means any group or compound that is allowed to exhibit its enzymatic activity. The group or compound is abasic in that it does not contain a generally recognized nucleotide base such as adenosine, guanine, cytosine, uracil or thymidine, for example at the C1 position of the sugar. May be.

  Synthesis of a chemically modifiable siNA molecule of the invention comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) under conditions suitable to obtain a double stranded siNA molecule, Annealing the two complementary strands together. In another embodiment, the synthesis of the two complementary strands of the siNA molecule is performed by solid phase oligonucleotide synthesis. In yet another embodiment, the synthesis of the two complementary strands of the siNA molecule is performed by solid phase tandem oligonucleotide synthesis.

  Oligonucleotides (eg, certain modified oligonucleotides, or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example, as described below: Caruthers et al. , Methods in Enzymology 211: 3-19, 1992; Thompson et al. , International Publication No. WO 99/54459; Wincott et al. , Nucleic Acids Res. 23: 2677-2684, 1995; Wincott et al. , Methods Mol. Bio. 74:59, 1997; Brennan et al. Biotechnol Bioeng. 61: 33-45, 1998; and Brennan, US Pat. No. 6,001,311. Synthesis of RNA comprising specific siNA molecules of the invention follows, for example, the general method described below: Usman et al. , J .; Am. Chem. Soc. 109: 7845, 1987; Scaringe et al. , Nucleic Acids Res. 18: 5433, 1990; and Wincott et al. , Nucleic Acids Res. 23: 2677-2684, 1995; Wincott et al. , Methods Mol. Bio. 74:59, 1997.

  Additional or complementary methods of delivery of nucleic acid molecules for use within the scope of the present invention are described, for example, in: Akhtar et al. , Trends Cell Bio. 2: 139, 1992; “Delivery Strategies for Antisense Oligonucleotide Therapeutics”, Akhtar, 1995; Maurer et al. Mol. Membr. Biol. 16: 129-140, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137: 165-192, 1999; and Lee et al. , ACS Symp. Ser. 752: 184-192, 2000. Sullivan et al. International Publication No. WO 94/02595 further describes general delivery methods for enzymatic nucleic acid molecules. These protocols can be utilized for additional or complementary delivery methods of virtually any nucleic acid molecule contemplated within the scope of the present invention.

  Nucleic acid molecules and polynucleotide delivery-enhancing polypeptides can be administered to cells by a variety of methods known to those skilled in the art, including a formulation comprising only siNA and a polynucleotide delivery-enhancing polypeptide, Or administration in a formulation further comprising one or more additional ingredients such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, etc. However, it is not limited to these. In certain embodiments, siNA and / or polynucleotide delivery-enhancing polypeptides can be encapsulated in liposomes, administered by iontophoresis, or hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres. Or can be incorporated into other media such as protein vectors (see, eg, O'Hare and Normand, International Publication No. WO 00/53722). Alternatively, the nucleic acid / peptide / vehicle combination can be delivered locally by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention can be performed using standard injection needle and syringe methods, whether subcutaneous, intramuscular, or intradermal, or by Conry et al., Clin. Cancer Res. 5: 2330-2337, 1999, and Barry et al., International Publication No. WO 99/31262, which can be performed by needle-free technology.

  The composition of the present invention can be effectively used as a pharmaceutical product. A medicinal product prevents or reduces the onset or severity of a medical condition or other adverse condition in a patient, or treats the medical condition (reduces one or more symptoms to a detectable or measurable extent). .

  Thus, in a further embodiment, the present invention is combined with or conjugated with a polynucleotide delivery-enhancing polypeptide that may be selectively formulated with a pharmaceutically acceptable carrier such as a diluent, stabilizer, buffer, etc. Provided are pharmaceutical compositions and methods characterized by the presence or administration of one or more polynucleic acids, typically one or more siNAs, embodied or bound.

  The present invention satisfies further challenges and advantages by providing small interfering nucleic acid (siNA) molecules that modulate the expression of genes associated with specific disease states or other deleterious conditions within a subject. Typically, siNA targets genes that are expressed at high levels as causative or contributing factors associated with a subject's condition or adverse condition. In this context, siNA will effectively downregulate the expression of the gene to a level that prevents, reduces or reduces the severity or recurrence of one or more associated disease symptoms. Alternatively, for a variety of different disease models where the expression of the target gene does not necessarily increase as a result or continuation of the disease or other deleterious condition, down-regulation of the target gene still reduces gene expression (i.e. Reducing the concentration of the selected mRNA and / or the protein product of the target gene will produce a therapeutic outcome. Alternatively, the siNA of the invention may be targeted to reduce the expression of one gene, thereby “downstream” whose expression is negatively controlled by the product or activity of the target gene. It is also possible to generate up-regulation of genes.

  In an exemplary embodiment, the compositions and methods of the present invention provide a treatment for modulating or expressing tumor necrosis factor-alpha (TNF-α) to treat or prevent symptoms of rheumatoid arthritis (RA). Useful as a tool. In this context, the present invention further provides compounds, compositions and methods useful for modulating TNF-α expression and activity by RNA interference (RNAi) using low molecular weight nucleic acid molecules. In more detailed embodiments, the present invention relates to small interfering nucleic acids (siNA), small interfering RNA (siRNA), double stranded RNA (dsRNA), microRNA (mRNA), short hairpin RNA (shRNA) molecules, etc. A low molecular weight nucleic acid molecule and a related method effective to regulate TNF-α and / or TNF-α gene expression to prevent or alleviate symptoms of RA in a mammalian subject. provide. In these and related therapeutic compositions and methods, the use of chemically modified siNA can e.g. provide for improved resistance to nuclease degradation in vivo and / or through improved cellular uptake of natural siNA molecules. Compared to properties, the properties of modified siNA will often be improved. Useful siNAs with multiple chemical modifications will retain their RNAi activity, as can be readily determined according to the disclosure herein. Thus, the siNA molecules of the present invention provide reagents and methods useful for various therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

  The siNA of the present invention can be administered, for example, transdermally or by local injection (eg, local injection at the site of psoriatic plaques to treat psoriasis, or local injection into the joints of patients suffering from psoriatic arthritis or RA). And can be administered in any form. In a more detailed embodiment, the present invention targets TNF-α mRNA and effectively reduces one or more TNF-α related inflammatory conditions by effectively down-regulating TNF-α RNA. Alternatively, formulations and methods for administering a therapeutically effective amount of siNA to prevent are provided. Comprising any of a number of genes that are known to be abnormally elevated in the expression of the gene as a causal factor or contributing factor associated with the selected medical condition in the animal subject, Similar methods and compositions targeting the expression of one or more different genes are also provided.

  The siNA / polynucleotide delivery-enhancing polypeptide mixtures of the present invention can be administered in conjunction with other standard treatments for the targeted disease state, for example, effective treatment for inflammatory diseases such as RA or psoriasis Can be administered with drugs. Examples of drugs that are useful and effective in combination in this context include nonsteroidal anti-inflammatory drugs (NSAIDs), methotrexate, gold compounds, D-penicillamine, antimalarials, sulfasalazine, glucocorticoids, and infliximab and entracept Other TNF-α neutralizing agents such as (entrept) are included.

  The negatively charged polynucleotide (eg, RNA or DNA) of the present invention may be administered to a patient by any standard technique in the presence or absence of stabilizers, buffers, etc. to form a pharmaceutical composition. it can. If it is desired to use a liposome delivery mechanism, standard protocols for forming liposomes can be followed. The compositions of the present invention include tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for administration by injection, and other compositions known in the art. It can be used as a preparation.

  The invention also encompasses pharmaceutically acceptable formulations of the compositions described herein. These formulations comprise salts of the above compounds, such as, for example, acid addition salts such as hydrochloric acid, hydrobromic acid, acetic acid, and salts of benzenesulfonic acid.

  A pharmacological composition or formulation refers to a composition or formulation that is in a form suitable for administration, eg, systemic administration, to a cell or patient, eg, a human. The appropriate form will depend in part on the method of use or route of entry, eg, oral, transdermal, or injection. Such a form does not prevent the composition or formulation from reaching the target cell (ie, the cell to which the negatively charged nucleic acid is desired to be delivered). For example, a pharmacological composition that is injected into the bloodstream must be soluble. Other factors are known in the art and include considerations such as toxicity.

  “Systemic administration” means systemic absorption or retention of a drug in the bloodstream in the bloodstream, followed by systemic dispersion. Routes of administration leading to systemic absorption include but are not limited to intravenous, subcutaneous, intraperitoneal, aspiration, oral, intrapulmonary and intramuscular. Each of these routes of administration exposes, for example, the desired negatively charged polymer, such as a nucleic acid, to reachable diseased tissue. The rate of drug entry into the circulatory system has been shown to be a function of molecular weight or size. The use of liposomes or other drug carriers comprising the compounds of the present invention allows for the potential localization of drugs into specific types of tissues, for example, reticuloendothelial system (RES) tissues. Become. For example, liposome preparations that can promote the association of drugs on the surface of cells such as lymphocytes and macrophages are also useful. This approach can provide improved drug delivery to target cells by exploiting the specificity of immune recognition of abnormal cells such as cancer cells by macrophages and lymphocytes.

  “Pharmaceutically acceptable formulation” means a composition or formulation that enables the nucleic acid molecule of the present invention to be effectively dispersed within a body site most suitable for its desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the present invention include the following: P-glycoprotein inhibitors (pluronic P85 () that can facilitate the entry of drugs into the central nervous system. (Pluronic P85), etc.) (Jolliet-Rient and Tillement, Fundam. Clin. Pharmacol. 13: 16-26, 1999); poly (DL-lactide-coglycolide) for sustained release after intracerebral transplantation Biodegradable polymers such as spheres (Emerich, DF et al., Cell Transplant 8: 47-58, 1999) (Alkermes, Inc., Cambridge, Mass.); And the blood brain barrier The delivery of drugs between is possible and God It is possible to change the uptake mechanism, made of polybutylcyanoacrylate, filled nanoparticles (loaded nanoparticle) (Prog Neuropsychopharmacol Biol Psychiatry 23: 941-949,1999). Other non-limiting examples of delivery means for the nucleic acid molecules of the invention include the materials described below: Boado et al. , J .; Pharm. Sci. 87: 1308-1315, 1998; Tyler et al. , FEBS Lett. 421: 280-284, 1999; Pardridge et al. , PNAS USA. 92: 5592-5596, 1995; Boado, Adv. Drug Delivery Rev. 15: 73-107, 1995; Aldrian-Herrada et al. , Nucleic Acids Res. 26: 4910-4916, 1998; and Tyler et al. , PNAS USA. . 96: 7053-7058, 1999.

  The present invention also includes compositions prepared for storage or administration that comprise a pharmaceutically effective amount of the desired compound in a pharmaceutically acceptable carrier or diluent. Carriers or diluents that are acceptable for therapeutic use are well known in the pharmaceutical art and are described, for example, in “Remington's Pharmaceutical Sciences”, Mack Publishing Co. ( Mack Publishing Co.), A.M. R. Edited by Gennaro, 1985. For example, preservatives, stabilizers, colorants and fragrances may be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition to the above, antioxidants and suspending agents can also be used.

  A pharmaceutically effective dose is the dose required to prevent, inhibit or treat the development of a medical condition (relieving some symptoms, preferably all symptoms to a certain extent). . The pharmaceutically effective dosage depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the particular mammal under consideration, the drugs used in combination, and the medical field. Depends on other factors recognized by those skilled in the art. Depending on the strength of action of the negatively charged polymer, the active ingredient is generally administered in an amount of 0.1 mg / kg to 100 mg / kg per kg of body weight per day.

  Aqueous suspensions contain the active ingredients in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are, for example, suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum; the dispersing or wetting agent is lecithin or stearin Condensates of alkylene oxides and fatty acids such as polyoxyethylene acid, or condensates of ethylene oxide and long-chain aliphatic alcohols such as heptadecaethyleneoxycetanol, or polyoxyethylene sorbitol monooleate (polyoxyethylene sorbitol monooleate) A condensate of ethylene oxide with a partial ester obtained from a fatty acid and hexitol, or polyethylene sorbitan monooleate Natural phosphatides such as a condensate of tylene oxide and a partial ester obtained from a fatty acid and hexitol anhydride may also be used. The aqueous suspension may include, for example, one or more preservatives, one or more colorants, one or more fragrances, such as ethyl or n-propyl p-hydroxybenzoate, and One or more sweeteners such as sucrose or saccharin may also be included.

  For example, an oily suspension can be prepared by suspending the active ingredient in a vegetable oil, such as peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspension may contain a thickener such as beeswax, hard paraffin or cetyl alcohol. Sweeteners and fragrances can be added to provide a palatable oral preparation. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

  Dispersible powders and granules suitable for preparing aqueous suspensions by the addition of water provide the active ingredient in a mixture with a dispersing or wetting agent, suspending agent and one or more preservatives To do. Suitable dispersing or wetting agents or suspending agents have already been mentioned and exemplified above. Additional excipients such as sweetening, flavoring and coloring agents may be present.

  The pharmaceutical composition of the present invention can also take the form of an oil-in-water emulsion. The oil reservoir may be vegetable oil or mineral oil or a mixture thereof. Suitable emulsifiers are natural rubbers such as gum acacia or tragacanth, for example natural phospholipids such as soy, lecithin, and esters or partial esters obtained from fatty acids and hexitol anhydrides such as sorbitan monooleate. And a condensate of the partial ester such as polyoxyethylene sorbitan monooleate with ethylene oxide. The emulsion may contain sweeteners and fragrances.

  The pharmaceutical compositions may take the form of a sterile injectable aqueous or oleagenous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation is a sterile injectable solution or solvent in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. It may be a suspension. Among the acceptable media and solvents, water, Ringer's solution and isotonic sodium chloride solution can be used, among others. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.

  siNA can also be administered, for example, in the form of suppositories for rectal administration of the drug. These compositions are obtained by mixing the drug with a suitable non-irritating excipient that is solid at ambient temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Can be prepared. Such materials include cocoa butter and polyethylene glycols.

  For example, siNA can be stably modified on a large scale by modification with a nuclease resistant group such as 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H, etc. It is possible to improve the property. For review, see: Usman and Cedergren, TIBS 17:34, 1992; Usman et al. Nucleic Acids Symp. Ser. 31: 163, 1994. siNA constructs can be purified by gel electrophoresis using general methods, or by high pressure liquid chromatography and resuspension in water.

  Chemically synthesized nucleic acid molecules with modifications (bases, sugars and / or phosphates) can be protected from degradation by ribonuclease in the serum and thus increase their potency. For an example, see: Eckstein et al. , International Publication No. WO 92/07065; Perrart et al. , Nature 344: 565, 1990; Pieken et al. , Science 253: 314, 1991; Usman and Cedergren, Trends in Biochem. Sci. 17: 334, 1992; Usman et al. , International Publication No. WO 93/15187; and Rossi et al. , International Publication No. WO 91/03162; Sproat, US Pat. No. 5,334,711; Gold et al. , US Pat. No. 6,300,074. All of the above references describe various chemical modification methods that can be performed 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 a nucleic acid molecule to significantly improve the stability and effectiveness of the nucleic acid molecule against nucleases. . For example, by modification with a nuclease resistance group such as 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modification, By modifying the oligonucleotide, stability is improved and / or biological activity is improved. For review, see: Usman and Cedergren, TIBS 17:34, 1992; Usman et al., Nucleic Acids Symp. Ser. 31: 163, 1994; Burgin et al. Biochemistry 35: 14090, 1996. In addition, modification of sugars of nucleic acid molecules has already been described in various ways in the technical field. See: Eckstein et al. , International Publication No. WO 92/07065; Perrart et al. , Nature 344: 565-568, 1990; Pieken et al. , Science 253: 314-317, 1991; Usman and Cedergren, Trends in Biochem. Sci. 17: 334-339, 1992; Usman et al. International Publication No. WO 93/15187; Sproat, US Pat. No. 5,334,711 and Beigelman et al. , J .; Biol. Chem. 270: 25702, 1995; Beigelman et al. , International Publication No. WO 97/26270; Beigelman et al. U.S. Pat. No. 5,716,824; Usman et al. U.S. Pat. No. 5,627,053; Woolf et al. , International Publication No. WO 98/13526; Thompson et al. Karpeskyy et al. Tetrahedron Lett. 39: 1131, 1998; Earnshow 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 to 2010, 1997. These publications describe general methods and means for determining the position to incorporate sugar, base and / or phosphate modifications, etc., into a nucleic acid molecule without altering the catalytic action. With reference to these techniques, similar modifications are used as described herein to the extent that siNA nucleic acid molecules of the invention do not significantly inhibit the ability of siNA to promote RNAi in cells. It is possible to modify.

  Chemical modification of the oligonucleotide's internucleotide linkages by phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate linkages improves stability, while over-modifications are of constant toxicity or activity. There is a risk of lowering. Thus, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. Decreasing the concentration of these bindings will reduce toxicity and consequently improve the effectiveness of these molecules and increase specificity.

  In one embodiment, the present invention relates to one or more of phosphorothioates, phosphorodithioates, methyl phosphonates, phosphotriesters, morpholinos, amidates, carbamates, carboxymethyl, acetamidate Featuring modified siNA molecules having modifications of the phosphate backbone with polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and / or alkylsilyl substitution. For a review of oligonucleotide backbone modifications, see: Hunziker and Leumann, “Nucleic acid analogs: Synthesis and Properties in Modern Synthesis Methods, in Modern Synthetic Methods, 19” V, 331-417 and Mesmaeker et al. , "Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research," 1994, pp. 24-39.

  Methods for delivery of nucleic acid molecules are described below: Akhtar et al. , Trends Cell Bio. 2: 139, 1992; “Delivery Strategies for Antisense Oligonucleotide Therapeutics”, Akhtar, 1995; Maurer et al. Mol. Membr. Biol. 16: 129-140, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137: 165-192, 1999; and Lee et al. , ACS Symp. Ser. 752: 184-192, 2000. Beigelman et al. U.S. Pat. No. 6,395,713 and Sullivan et al. International Publication No. WO 94/02595 further describes general delivery methods for nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules include, but are not limited to, encapsulation in liposomes, iontophoresis, or biodegradable polymers, hydrogels, cyclodextrins (for example, Gonzalez et al., Bioconjugate Chem. 10: 1068-1074, 1999; Wang et al., See International Publication Nos. WO03 / 47518 and WO03 / 46185), poly (lactic-co-glycolic acid) (PLGA). And other media, such as PLCA microspheres (see, for example, US Pat. No. 6,447,796 and US Patent Publication No. US2002130430), biodegradable nanocapsules, and bioadhesive microspheres Uptake or protein Can be administered to cells by a variety of methods known to those of skill in the art, including: Kuta (O'Hare and Normand, International Publication No. WO 00/53722). Alternatively, in the alternative, the nucleic acid / vehicle combination is locally delivered by direct injection or use of an infusion pump. Direct injection of the nucleic acid molecules of the invention can be performed using standard injection needle and syringe methods, whether subcutaneous, intramuscular, or intradermal, or by Conry et al., Clin. Cancer Res. 5: 2330 to 2337, 1999, and Barry et al., International Publication No. WO 99/31262, which can be performed by needle-free technology. The molecules of the present invention can be used as pharmaceuticals. A medicinal agent prevents and relieves or treats the onset of a medical condition within a patient (a certain symptom, preferably alleviates all symptoms to a certain extent).

  The term “ligand” is any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that can interact directly or indirectly with another compound, such as a receptor. Or it refers to a molecule. The receptor that interacts with the ligand may be present on the surface of the cell or, alternatively, may be an intracellular receptor. Interaction with a receptor by a ligand can cause a biochemical reaction, or it can be just a physical interaction or association.

  As used herein, an “asymmetric hairpin” is a linear siNA molecule comprising an antisense region, a loop portion that may comprise nucleotides and non-nucleotides, and a sense region, wherein the sense region comprises Means a siNA molecule comprising fewer nucleotides than the antisense region, as long as it has sufficient complementary nucleotides to base pair with the antisense region to form a duplex with the loop To do. By way of example, an asymmetric hairpin siNA molecule of the invention is long enough to mediate RNAi in a T cell, eg, about 19 to about 22 (eg, about 19, 20, 21, or 22) nucleotides. An antisense region having a length and a loop region comprising about 4 to about 8 (eg, about 4, 5, 6, 7, or 8) nucleotides, and about 3 to about 18 complementary to said antisense region ( For example, it may comprise a sense region having about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides. The asymmetric hairpin siNA molecule can also comprise a chemically modifiable 5 'terminal phosphate group. The loop portion of the asymmetric hairpin siNA molecule can comprise a nucleotide, non-nucleotide, linker molecule, or conjugate molecule as described herein.

  As used herein, “asymmetric duplex” refers to a siNA molecule having two separate strands comprising a sense region and an antisense region, where the sense region is the antisense region and Insofar as it has sufficient complementary nucleotides to form a base pair to form a duplex, it comprises fewer nucleotides than the antisense region. By way of example, an asymmetric duplex siNA molecule of the invention is sufficient to mediate RNAi in T cells, such as, for example, about 19 to about 22 (eg, about 19, 20, 21, or 22) nucleotides. And about 3 to about 18 complementary to the antisense region (eg, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). , 15, 16, 17 or 18) can comprise a sense region having nucleotides.

  “Modulate gene expression” means that the expression of a target gene is up-regulated or down-regulated, and the intracellular mRNA concentration, or translation of mRNA, or encoded in the target gene Up-regulation or down-regulation of the synthesis of the protein or protein subunit to be carried out. Modulation of gene expression is such that the expression, concentration or activity of one or more proteins or protein subunits encoded by up- or down-regulated target genes is in the absence of a modulator (eg, siRNA). It can also be measured by the presence, amount or activity of the protein or subunit of interest, such as whether it is higher or lower than observed. For example, the term “modulate” may mean “inhibit,” but the use of the word “modulate” is limited to this definition. is not.

  “Inhibit”, “down-regulate”, or “reduce” expression is the expression of a gene, or an RNA molecule or one or more proteins or protein sub- The concentration of the equivalent RNA molecule encoding the unit, or the concentration or activity of one or more proteins or protein subunits encoded by the target gene is determined in the absence of the nucleic acid molecule of the invention (eg, siNA). Means lower than observed. In one embodiment, inhibition, down-regulation or reduction by siNA molecules is below the level observed in the presence of inactive or attenuated molecules. In another embodiment, inhibition, down-regulation, or reduction by siNA molecules is below the level observed in the presence of, for example, messy sequences or siNA molecules with mismatches. In another embodiment, inhibition, down-regulation or reduction of gene expression using the nucleic acid molecules of the invention is greater in the presence of the nucleic acid molecule than in the absence.

  Gene “silencing” refers to partial or complete loss-of-function due to targeted inhibition of gene expression in a cell, and is referred to as “knock down”. Sometimes called. Depending on the situation and the biological challenge to be addressed, it may be desirable to partially reduce gene expression. In the alternative, it would be desirable to reduce gene expression as much as possible. The degree of silencing can be measured by methods known in the art, some of which are summarized in International Publication No. WO 99/32619. Depending on the assay, quantification of gene expression allows detection of the various amounts of inhibition desired in certain embodiments of the invention, which include mRNA concentration or protein concentration or For activity, for example, baseline (ie, normal value) or other control levels with high expression levels that may be associated with the particular condition or other disorder being treated, or these values 10%, 30%, 50%, 75%, 90%, 95%, or 99% of the above include prophylaxis and treatment methods capable of knocking down the expression of the target gene.

  The phrase “inhibiting expression of a target gene” refers to the ability of the siNA of the present invention to initiate gene silencing of a target gene. To examine the extent of gene silencing, a sample or assay of cells in culture or a subject organism that expresses a particular construct is compared to a control sample that does not express the construct. The control sample (not expressing the construct) is defined as a relative value of 100%. Inhibition of target gene expression has been achieved when the test value relative to the control is about 90%, often 50%, and in certain embodiments 25-0%. Appropriate assays include techniques known to those skilled in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function methods, and expressions known to those skilled in the art. Examples include protein or mRNA concentration testing by type analysis.

  A “subject” is an organism, tissue, or cell that will comprise a subject, or an organism as a donor or recipient of cells to be transplanted, or a cell that is itself the subject of siNA delivery. Means. Thus, a “subject” is an in vitro or ex vivo such that a nucleic acid molecule of the invention can be administered and the nucleic acid molecule can be enhanced therein by a polynucleotide delivery-enhancing polypeptide as described herein. May refer to an organism, organ, tissue, or cell comprising a subject of any organ, tissue, or cell. Exemplary subjects include, for example, mammalian individuals or cells, such as human patients or cells.

  As used herein, “cell” is used in its normal biological sense and does not refer to an entire multicellular organism, for example, specifically to a human. The cells may be present in birds, plants, and living organisms such as mammals such as humans, cows, sheep, apes, monkeys, pigs, dogs, and cats. The cell may be a prokaryotic cell (for example, a bacterial cell) or a eukaryotic cell (for example, a mammalian cell or a plant cell). Further, the cell may be a somatic cell-derived cell or a germ cell-derived cell, a totipotent cell or other brain cell, a dividing cell, or a non-dividing cell. The cells may be gametes or embryos, stem cells, cells derived from fully differentiated cells, or cells comprising these.

  By “vector” is meant any nucleic acid and / or virus-based technique used to deliver a desired nucleic acid.

  “Comprising” means including, but not limited to, anything following the word “comprising”. Thus, the use of the term “comprising” means that the listed elements are necessary or required, but other elements are optional and are present but not present. Indicates that it may be. “Consisting of” means encompassing and limited to anything following the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are necessary or required, and that no other elements should be present. “Consisting essentially of” comprises any element listed after the phrase, but does not interfere with the activity or action specified in the description of the listed element, or It means that it is limited to other elements that do not contribute to. Thus, the phrase “consisting essentially of” means that the listed elements are required or required, but other elements are optional, and Indicates that it may or may not be present depending on whether it affects activity or action.

  “RNA” means a molecule comprising at least one ribonucleotide residue. “Ribonucleotide” means a nucleotide having a hydroxyl group at the 2 ′ position of a β-D-ribofuranose moiety. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, RNA made by recombinant technology, As well as modified RNA by addition, deletion, substitution and / or modification of one or more nucleotides different from the natural RNA. Such alterations include, for example, addition of non-nucleotide material to the end of siNA or to one or more nucleotides within the RNA. The nucleotides in the RNA molecules of the present invention may comprise non-natural nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs or analogs of natural RNA.

  “Highly conserved sequence region” means that the nucleotide sequence of one or more regions in a target gene is transferred from one generation to the next, or from one biological system to another. It means that there is no significant change across the biological system.

  “Sense region” means the nucleotide sequence of a siNA molecule having complementarity with the antisense region of the siNA molecule. In addition, the sense region of the siNA molecule may include a nucleic acid sequence having homology with the target nucleic acid sequence.

  “Antisense region” means the nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of the siNA molecule may optionally include a nucleic acid sequence having complementarity with the sense region of the siNA molecule.

  “Target nucleic acid” means any nucleic acid sequence whose expression or activity is regulated. The target nucleic acid may be DNA or RNA.

  “Complementarity” means that a nucleic acid can form a hydrogen bond with another nucleic acid sequence in a conventional Watson-Crick or other non-conventional manner. For the nucleic acid molecules of the present invention, the free energy of binding of the nucleic acid molecule to its complementary sequence is sufficient to trigger the appropriate function of the nucleic acid, such as RNAi activity. Measurement of free energy of binding of nucleic acid molecules is well known in the art (see, for example, Turner et al., CSH Symp. Quant. Biol., LII, 1987, pp. 123-133; Frier; Et al., Proc. Nat. Acad. Sci. USA 83: 9373-9377, 1986; Turner et al., J. Am.Chem.Soc. 109: 3783-3785, 1987). The percentage of complementarity indicates the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (eg, Watson-Crick base pairs) with a second nucleic acid sequence (eg, a first oligonucleotide). Out of a total of 10 nucleotides, 5, 6, 7, 8, 9, or 10 nucleotides form a base pair with a second nucleic acid sequence having 10 nucleotides, respectively, 50%, 60%, 70%, 80%, 90% and 100% complementarity). “Perfectly complementary” means that all contiguous residues of a nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence.

  As used herein, the term “universal base” refers to analogs of nucleotide bases that form base pairs with each of the natural DNA / RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole, carboxamide, and 3-nitropyrrole, 4-nitroindole, 5- Nitroindoles and nitroazole derivatives such as 6-nitroindole are included (see, for example, Loakes, Nucleic Acids Research 29: 2437-2447, 2001).

  As used herein, the term “acyclic nucleotide” refers to, for example, any of the ribose carbons (C1, C2, C3, C4, or C5) present in a nucleotide independently or in combination. Refers to any nucleotide having an acyclic ribose sugar, such as

  As used herein, the term “biodegradable” refers to degradation in a biological system such as, for example, enzymatic degradation or chemical degradation.

  The term “biologically active molecule” as used herein refers to a compound or molecule capable of inducing or altering a biological response in a system. Non-limiting examples of biologically active siNA molecules alone or in combination with other molecules contemplated by the present invention include antibodies, cholesterol, hormones, antiviral agents, peptides, proteins, chemotherapeutic agents, small molecules, vitamins, Therapeutic activity such as cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys, and analogs thereof Includes molecules. The bioactive molecules of the present invention can alter the pharmacokinetics and / or pharmacodynamics of other bioactive molecules such as lipids and polymers such as polyamines, polyamides, polyethylene glycols and other polyethers. Also includes molecules.

  As used herein, the term “phospholipid” refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid contains a phosphorus-containing group and a saturated or unsaturated alkyl group, and may be optionally substituted with OH, COOH, oxo, amine, or substituted or substituted with an aryl group It does not have to be.

  “Cap structure” means a chemical modification incorporated at either end of an oligonucleotide (eg, Adamic et al., US Pat. No. 5,998, incorporated herein by reference). , 203). These terminal modifications can protect the nucleic acid molecule from degradation by exonucleases and assist in delivery and / or localization into the cell. The cap may be present at the 5 'end (5' cap), the 3 'end (3' cap), or may be present at both ends. In a non-limiting example, the 5 ′ cap includes, but is not limited to: glyceryl, inverted deoxy abasic residue (moiety); 4 ′, 5′-methylene Nucleotides; 1- (β-D-erythrofuranosyl) nucleotides, 4′-thionucleotides; carbocyclic nucleotides; 1,5-anhydrohexitol nucleotides; L-nucleotides; α-nucleotides; Rothioate linkage; threo-pentofuranosyl nucleotide; acyclic 3 ′, 4′-seconucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3 '-Inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted 1,4-butanediol phosphate; 3'-phosphoramidate; hexyl phosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; Or a cross-linked or non-cross-linked methyl phosphonate moiety.

  Non-limiting examples of 3 'caps include, but are not limited to: glyceryl, inverted deoxy abasic residue (moiety); 4', 5'-methylene nucleotides 1- (β-D-erythrofuranosyl) nucleotide, 4′-thionucleotide, carbocyclic nucleotide; 5′-aminoalkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotides; L-nucleotides; α-nucleotides; modified base nucleotides; Binding; threo-pentofuranosyl nucleotide; acyclic 3 ′, 4′-seconucleotide; 3 4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridged and / or unbridged 5′-phosphoramidate, phosphorothioate and / or phosphorodithioate, bridged or unbridged methylphosphonate And 5 ′ mercapto moieties (for details see Beaucage and Lyer, Tetrahedron 49: 1925, 1993, which is incorporated herein by reference).

  The term “non-nucleotide” can be incorporated into a nucleic acid strand in place of one or more nucleotide units, including any substitution by sugar and / or phosphate, and the remaining base Refers to any group or compound capable of exhibiting its enzymatic activity. The groups and compounds are abasic in that they do not contain commonly recognized nucleotide bases such as adenosine, guanine, cytosine, uracil or thymine, and thus lack a base at the 1 'position.

  The term “nucleotide” as used herein includes natural bases (standards) and modified bases well known in the art, as recognized in the art. Such a base is generally present at the 1 'position of the nucleotide sugar moiety. In general, a nucleotide comprises a base, a sugar and a phosphate group. The nucleotide may be unmodified or modified in sugar, phosphate and / or base moieties (alternately called nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others) See, for example: Usman and McSwiggen, supra; Eckstein et al., International Publication No. WO 92/07065; Usman et al., International Publication No. WO 93/15187; Uhlman and Peyman, supra: All of which are incorporated herein by reference). Examples of multiple modified nucleobases are known in the art and are summarized below: Limbach et al. , Nucleic Acids Res. 22: 2183, 1994. Non-limiting examples of base modifications that can be introduced into a nucleic acid molecule include: inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (eg, 5-methylcytidine), 5-alkyluridine (eg, ribothymidine), 5-halouridine (eg, 5-bromouridine) or 6-azapyrimidine or 6-alkylpyridine (eg 6-methyluridine), propyne, and others (Burgin et al., Biochemistry 35: 1409, 1996; Uhlman and Peyman, supra). “Modified base” in this embodiment means a nucleotide base other than adenine, guanine, cytosine and uracil at position 1 ′, or equivalents thereof.

  “Target site” means a sequence in the target RNA that is the “target” of cleavage mediated by the siNA construct comprising within its antisense region a sequence complementary to the target sequence.

  “Detectable level of cleavage” is the degradation of target RNA (and the cleavage product RNA to the extent that the degradation products above the background of the RNA created by random degradation of the target RNA can be fully confirmed). Formation). In most detection methods, production of 1-5% degradation products of the target RNA is detected well above background.

  A “biological system” is in a purified or unpurified form derived from a biological source including, but not limited to, human, animal, plant, insect, bacterial, viral or other sources. Means a substance, in which case the system comprises components necessary for RNAi activity. The term “biological system” includes, for example, cells, tissues, or organisms, or extracts thereof. The term biological system also includes a reconstituted RNAi system that can be used in an in vitro environment.

  As used herein, the term “biodegradable linker” refers to, for example, linking a biologically active molecule to a siNA molecule of the invention or connecting the sense and antisense strands of a siNA molecule of the invention. It refers to a nucleic acid or non-nucleic acid linker molecule designed as a biodegradable linker for linking one molecule to be linked to another molecule. The biodegradable linker is designed such that its stability can be adjusted for special purposes, such as delivery to a particular type of tissue or cell. The stability of nucleic acid-based biodegradable linker molecules is e.g. ribonucleotides, deoxyribonucleotides, as well as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C. It can be modulated using a variety of chemistries such as combinations of chemically modified nucleotides such as -allyl, 2'-O-allyl, and other 2'-modified or base-modified nucleotides. The biodegradable nucleic acid linker molecule is a dimer, trimer, tetramer, or longer nucleic acid molecule, eg, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, It may be an oligonucleotide of 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or a phosphorus-based linkage such as, for example, a phosphoramidate or phosphodiester linkage It may contain a single nucleotide having The biodegradable nucleic acid linker molecule may also include a nucleic acid backbone, nucleic acid sugar, or nucleic acid based modification.

  “Abasic” means a sugar moiety that lacks a base or has other chemical groups in place of the base at the 1 ′ position: see, for example, Adamic et al. , U.S. Pat. No. 5,998,203.

  “Unmodified nucleoside” means one of the bases of adenine, cytosine, guanine, thymine, or uracil attached to the 1 ′ carbon of β-D-ribofuranose.

  "Modified nucleoside" means any nucleotide base that comprises a modification in the chemical structure of an unmodified nucleotide base, sugar and / or phosphate. Non-limiting examples of modified nucleotides are illustrated by Formulas I-VII and / or other modifications described herein.

In the context of the 2 ′ modified nucleotides described for the present invention, “amino” refers to 2′-NH ₂ or 2′-0--NH _2, which may be modified or unmodified. means. Such modifying groups are described, for example, in: Eckstein et al. U.S. Pat. No. 5,672,695 and Mathic-Adamic et al. , US Pat. No. 6,248,878.

  siNA molecules can be complexed with cationic lipids, encapsulated in liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complex can be locally administered through injections, infusion pumps or stents, with or without incorporation into the biopolymer. In another embodiment, polyethylene glycol (PEG) can be covalently attached to the siNA compounds of the invention, polynucleotide delivery-enhancing polypeptides, or both. The PEG attached may be of any molecular weight, but is preferably from about 2,000 to about 50,000 daltons (Da).

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

  “Inverted repeat” means that when the repeat is transcribed, the sense element and the antisense element are arranged so that the sense element and the antisense element can form a double-stranded siRNA. Refers to a nucleic acid sequence comprising. The inverted repeat may optionally contain a heterologous sequence such as a linker or a self-cleaving ribozyme between the two elements of the repeat. The elements of the inverted repeat have a length sufficient to form double stranded RNA. Typically, each element of the inverted repeat is about 15 to about 100 nucleotides in length, preferably about 20 to 30 bases nucleotides, preferably about 20 to 25 nucleotides in length. For example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

  “Nucleic acid” refers to deoxyribonucleotides, or ribonucleotides, and these polymers in single- or double-stranded form. The term includes synthetic, natural, and non-natural nucleic acids with known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid and Nucleic acids that are metabolized as well as nucleotides are included. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). It is not something.

  “Large double-stranded RNA” refers to any double-stranded RNA having a size greater than about 40 base pairs (bp), for example greater than 100 bp or more particularly , Refers to double-stranded RNA exceeding 300 bp. The sequence of a giant dsRNA may represent a segment of mRNA or the entire mRNA. The maximum size of the giant dsRNA is not limited here. The double-stranded RNA may contain a modified base, in which case the modification may be on the phosphate sugar backbone or nucleoside. Such modifications may include nitrogen or sulfur heteroatoms, or any other modification known in the art.

  Double stranded structures can be formed by self-complementary RNA strands such as those found in hairpin RNAs or microRNAs, or by annealing two different complementary RNA strands.

  “Overlapping” refers to the case where two RNA fragments have a sequence in which a plurality of nucleotides overlap on one strand. For example, in this case, the number of the plurality of nucleotides (nt) is: Only 2 to 5 nucleotides, or 5 to 10 nucleotides or more.

  “One or more dsRNAs” refer to dsRNAs that differ from each other in 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 can be representative of the target. Target genes or mRNAs can include genes involved in development and regulatory genes, as well as metabolic or structural genes, or genes encoding enzymes. The target gene may be expressed in the cell whose phenotype is the subject of study, or may be expressed in a living organism so as to directly or indirectly affect the phenotypic characteristics. The target gene may be endogenous or exogenous. Such cells include any cell with gametes in the body of an animal or plant that is an adult or embryo, or any isolated cell such as found in immortalized cell lines or primary cell cultures. It is.

  In this specification and the appended claims, the singular forms “a”, “an”, and “the” refer to the plural subject unless the context clearly dictates otherwise. Are also included.

EXAMPLES While the above disclosure generally describes the present invention, the present invention is further illustrated by the following examples. These examples are described for purposes of illustration only and are not intended to limit the scope of the invention. Although specific terms and numerical values are used in the present embodiment, these terms and numerical values are also understood to be exemplary rather than limiting the scope of the present invention. Should.

Example 1
Preparation and characterization of a composition comprising siRNA complexed with a polynucleotide delivery-enhancing polypeptide To form a complex between a candidate siRNA and a polynucleotide delivery-enhancing polypeptide of the invention, eg, OptiMEM (Opti-MEM) (registered trademark) cell culture medium (Invitrogen) or the like, an appropriate amount of siRNA is mixed with a predetermined amount of polynucleotide delivery-enhancing polypeptide at a predetermined ratio, and about 10 to about 10 at room temperature. Incubate for 30 minutes. Subsequently, for example, a selected volume of the mixture, such as about 50 μl, is contacted with the target cells and the cells are incubated for a predetermined incubation time, which in this example was about 2 hours. The siRNA / peptide mixture may optionally contain other additives such as cell culture medium or fetal calf serum. For H3, H4 and H2b, a series of experiments were performed to complex these polynucleotide delivery-enhancing polypeptides with siRNA at different ratios. Typically, this complexation was initiated with a siRNA / histone ratio of 1: 0.01 to 1:50. 40 pm siRNA was added to each well of a 96 well microtiter plate. Each well contained 50% confluent β-gal cells. Typical ratios optimized for transfection efficiency are shown in Table 2 below.

  9L / β-gal cells were transfected with standard siRNA or siRNA complexed with one of the histone proteins identified above. The siRNA is designed to specifically knock down β-galactosidase mRNA and the activity is expressed as a percentage of β-gal activity from the control (control cells are polynucleotide delivery enhancing polypeptides). Transfected with Lipofectamine without).

  Detection and / or quantitative assays of siRNA delivery efficiency are performed by conventional methods such as, for example, β-galactosidase assay or flow cytometry.

For the β-galactosidase assay, 9L / LacZ cells, which are cell lines that constantly express β-galactosidase, were used. 9L / LacZ cells are rat gliosarcoma fibroblasts that constitutively express LacZ and were obtained from ATCC (# CRL-2200). 9L / LacZ cells were grown in medium in Dulbecco's Modified Essential Medium (DMEM) supplemented with 1 mM sodium pyruvate, non-essential amino acids, and 20% fetal bovine serum. Cells are cultured at 37 ° C. with 5% CO ₂ supplemented with an antibiotic mixture containing 100 units / ml penicillin, 100 μg / ml streptomycin and 0.25 mg / ml Fungizone (Invitrogen) did. 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 a CLEAR-amide resin using a Rainin Symphony synthesizer. The coupling step was carried out over 40 minutes using 5 equivalents of HCTU and Fmoc amino acid with an excess of N-methylmorpholine. The peptide resin was treated twice with 20% piperidine in DMF for a period of 10 minutes to achieve Fmoc elimination. After completion of the entire peptide, the Fmoc group was removed using piperidine and washed thoroughly with DMF. Maleimide modified peptides were 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 degree of coupling was monitored by the Kaiser test. The peptide was cleaved from the resin by the addition of 10 mL of TFA with 2.5% water and 2.5 triisopropylsilane, and then gently stirred for 2 hours at room temperature. The resulting crude peptide was recovered by trituration with ether followed by filtration. The crude product was dissolved in Millipore water and lyophilized. The crude peptide was dissolved in 15 mL water and 3 mL acetic acid containing 0.05% TFA and reverse phase Zorbax RX-C8 (22 mm diameter) at a flow rate of 5 mL / min through a 5 mL injection loop. × 250 mm, particle size 5 μm). Purification was achieved by a linear AB gradient of 0.1% B / min where solvent A is 0.05% TFA in water and solvent B is 0.05% TFA in acetonitrile. The purified peptide was analyzed by HPLC and ESMS.

Synthesis and Preparation of siRNA Oligonucleotide synthesis can be performed using the optimal 5′-O-dimethyltrityl-2′-Ot-butyldimethylsilyl-3′-O-succinylribonucleoside, or 5 ′ where applicable. On a controlled porous glass of long-chain alkylamine derivatized from —O-dimethyltrityl-2′-deoxy-3′-0-succinylthymidine support, a standard 2-cyanoethylphospho Performed by the loamidite method. All oligonucleotides were synthesized on an ABI 3400 DNA / RNA synthesizer at either 0.2 or 1 μmol scale, cleaved from the solid support using concentrated NH ₄ OH, and NH ₄ OH: EtOH. Deprotect using a 3: 1 mixture at 55 ° C. The deprotection of the 2′-TBDMS protecting group is carried out by converting a base-deprotected RNA (N-methylpyrrolidone / triethylamine / triethylamine tris (hydrofluoride) (volume ratio 6: 3: 4) at 65 ° C. This was accomplished by incubating with a solution of (600 μL per μmol) for 2.5 hours. Components corresponding to A, U, C and G, 5'-dimethoxytrityl-N- (tac) -2'-O- (t-butyldimethylsilyl) -3 '-[(2-cyanoethyl)-(N , N-diisopropyl)]-phosphoramidite (Proligo, Boulder, Colorado), and the modified phosphoramidite 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 the supplier. Triethylamine trihydrofluoride, N-methylpyrrolidinone and concentrated ammonium hydroxide were purchased from Aldrich. All HPLC analyzes and purifications were performed using a Waters 2690 equipped with an Xterra ™ column. All other reagents are available from Glen Research Inc. Purchased from the company. The oligonucleotide was purified to a purity of over 97% as measured by RP-HPLC. SiRNA for mouse injection was purchased from Qiagen, and after annealing the siRNA was purified by HPLC to an endotoxin concentration acceptable for in vivo injection.

Cell culture
Primary human monocytes:
Fresh human blood samples from healthy donors were purchased from Golden West Biologicals. To isolate monocytes, immediately after receipt, blood samples were diluted 1: 1 with PBS. Peripheral blood mononuclear cells (PBMC) were first isolated from whole blood by a Ficoll (Amersham) gradient. Monocytes were further purified from PBMCs using Miltenyi CD14 positive selection kit and supplied protocol (MILTENYI BIOTEC). To assess the purity of monocyte samples, cells were incubated with anti-CD14 antibody (BD Biosciences) and then sorted by flow cytometry. The purity of the monocyte sample was over 95%.

  0.1 to 1.0 ng / ml liposaccharide (LPS) (Sigma, St. Louis, MO) was added to the cell culture to promote production of tumor necrosis factor- ± (TNF- ±). Activation of the sphere was performed. Cells were harvested after 3 hours of incubation with LPS, and mRNA concentrations were measured using the Quantine assay (Genospectra, Fremont, Calif.) According to the manufacturer's instructions.

Mouse tail fibroblasts:
Mouse tail fibroblasts (MTF) were obtained from the tail of C57BL / 6J mice. The tail was cut off and soaked in 70% ethanol, then chopped into small pieces with a razor blade. Fragments were washed 3 times with PBS and the tissue was disrupted by incubation in a shaker with 0.5 mg / mL collagenase, 100 units / mL penicillin and 100 μg / mL streptomycin at 37 ° C. Tail fragments were then removed from complete tail of medium (Dulbecco's modified essential medium (Dulbecco supplemented with 20% FBS, 1 mM sodium pyruvate, non-essential amino acids and 100 units / mL penicillin and 100 μg / mL streptomycin) until cells were established. 's Modified Essential Medium)). Cells were cultured at 37 ° C., 5% CO ₂ in the complete medium outlined above.

On the first day of the transfection procedure , remove the saturated 9L / LacZ culture from the T75 flask, detach the cells and remove 10 ml complete medium (DMEM, 1 × PS, 1 × sodium pyruvate, 1 × NEAA). Dilute in). The cells are further diluted to 1:15 and 100 μl of this sample is dispensed into a 96-well plate, which usually gives approximately 50% cell confluency the next day for transfection. become. Leave the wells at the periphery of the plate empty, fill with 250 μl of water, and leave the plate in the incubator at 37 ° C. (5% CO ₂ incubator) overnight, without stacking.

On the second day, transfection complexes are prepared in 50 [mu] l Opti-MEM per well. Remove the media from the plate and wash each well once with 200 μl PBS or OptiMEM. Invert the plate and use tissue paper to absorb moisture and dry completely. The transfection mixture is then added to each well (50 μl / well) and 250 μl of water is added to the peripheral wells to prevent them from drying. The cells are then incubated for at least 3 hours at 37 ° C. (5% CO ₂ incubator). Remove the transfection mixture and replace with 100 μl complete medium (DMEM, 1 × PS, 1 × sodium pyruvate, 1 × NEAA). Cells are cultured for a predetermined length of time and then harvested for enzyme assay.

Cell viability (MTT assay)
Cell viability will be assessed by MTT assay (MTT-100, MatTek kit). This kit measures the uptake of tetrazolium salt and the change to formazan dye. One hour prior to the end of the dosing time, 2 mL of MTT concentrate is mixed with 8 mL of MTT diluent and a lipid is used to prepare a melt diluted MTT concentrate. Each cell culture insert is washed twice with PBS containing Ca ⁺² and Mg ⁺² and then transferred to a new 96 well transport plate with 100 μL of mixed MTT solution per well. The 96 well transport plate is then incubated for 3 hours at 37 ° C., 5% CO ₂ . After a 3 hour incubation, the MTT solution is removed and the culture is transferred to a second 96 well feeder tray with 250 μL of MTT extraction solution per well. An additional 150 μL of MTT extraction solution was added to the surface of each culture well, and this sample was allowed to stand in a dark room at room temperature for a minimum of 2 hours to a maximum of 24 hours. The insert membrane was then pierced with a pipette tip to mix the top and bottom solutions of the well. Along with the extracted blank (negative control), 200 microliters of the mixed extraction solution was transferred to a 96 well plate for measurement in a microplate reader. The absorbance (OD) of the sample was measured at 570 nm by background subtraction at 650 nm using a plate reader. Cell viability was expressed as a percentage and was calculated by dividing the OD reading of the treated insert by the OD reading of the PBS-treated insert and multiplying this by 100. In this assay, it was assumed that PBS had no effect on cell viability and therefore showed 100% cell viability.

Enzyme Assay Reagents for enzyme assay are from Invitrogen (β-Gal Assay Kit (β-Gal Assay Kit)) and Fisher (Pierce Micro BCA Protein Assay Reagent Kit, catalog) Purchased.
A: Cell lysis • Remove media, wash once with 200 μl PBS, invert plate and blot dry.
Add 30 μl of lysis buffer from the β-Gal kit to each well.
• Freeze-thaw cells twice to create lysate.
B: β-Gal assay Prepare assay mix (50 μl 1 × buffer, 17 μl ONPG in each well).
Remove new plate and add 65 μl assay mix to each well.
Add 10 μl of cell lysate to each well. Blank wells must be provided for subtraction of background activity.
Incubate at 37 ° C for about 20 minutes, but long incubations should be avoided as they use up ONPG and bias high expression.
Add 100 μl stop solution.
• Measure OD at 420 nm.
C: BCA assay • Prepare a BSA standard (150 ul in each well), but duplicate each point on each plate.
• Place 145 μl of water in each well and add 5 μl of cell lysate to each well.
Prepare the final assay reagent (Assay Reagent) according to the manufacturer's instructions.
Add 150 μl assay reagent (Assay Reagent) to each well.
Incubate at 37 ° C for about 20 minutes.
• Measure OD at 562 nm.
D: Calculation of specific activity Specific activity is expressed in nmol / t / mg protein of hydrolyzed ONPG, where t is the incubation time in minutes at 37 ° C; The protein to be assayed measured by

Measurement of FITC / FAM-binding siRNA by flow cytometry Fluorescence activated cell sorting (FACS) using Beckman Coulter FC500 cell analyzer (Fullerton, Calif.) Analysis was carried out. The instrument was conditioned according to the fluorescent probe used (FAM or Cy5 for siRNA and FITC and PE for CD14). Propidium iodide (Fluka, St. Louis, MO) and Annexin V (R & D systems, Minneapolis, MN) were used as indicators of cell viability and cytotoxicity. A brief step-by-step protocol is detailed below.
a) After exposure to the siRNA / peptide complex, the cells were incubated for at least 3 hours.
b) Wash cells with 200 μl PBS.
c) The cells are detached with 15 μl TE and incubated at 37 ° C.
d) Resuspend cells in 5 wells with 30 μl FACS solution (PBS with 0.5% BSA and 0.1% sodium azide).
e) Combine all five wells into one tube.
f) Add 5 μl of PI (propidium iodide) to each tube.
g) Analyze cells by fluorescence activated cell sorting (FACS) according to manufacturer's instructions.

The siRNA sequences used for silencing β-galactosidase mRNA are as follows:
C. U. A. C. A. C. A. A. A. U. C. A. G. C. G. A. U. U. U. DT. DT (sense) (SEQ ID NO: 32)
A. A. A. U. C. G. C. U. G. A. U. U. U. G. U. G. U. A. G. dT. dT (antisense) (SEQ ID NO: 33)

  The data for this example is shown in Table 2. Transfection efficiency is negatively correlated with the amount of β-galactosidase activity measured from cell lysates. A decrease in β-galactosidase activity measured after transfection indicates successful transfection. Thus, if no transfection has occurred, the measured β-galactosidase activity is 100% and the transfection efficiency is 0%. The lower the β-galactosidase activity, the higher the transfection efficiency. For example, Table 2 shows that histone H2B and siRNA produced a transfection efficiency of 62.03% and the measured β-galactosidase activity was reduced to 37.97%. For the data shown in Table 3, the same method for measuring transfection efficiency was used.

Table 2:

To evaluate the efficiency of addition of cationic lipids to siRNA / peptide / lipid siRNA / polynucleotide delivery-enhancing polypeptide mixtures, complexes or conjugates, the lipofectamine at a constant concentration in the siRNA / polynucleotide delivery formulation according to the manufacturer's instructions The above procedure was followed except that (Invitrogen) was added (0.2 μl / OptiMEM 100 μl).

  To make a composition comprising GKINLKALAALAKIL (SEQ ID NO: 28), siRNA and Lipofectin® (Invitrogen), siRNA and peptide were first mixed together in Opti MEM cell culture medium at room temperature. Thereafter, Lipofectin® was added to the mixture at room temperature to form a composition of siRNA / peptide / cationic lipid composition.

  To make a composition comprising RVIRVWFQNKRCKDKK (SEQ ID NO: 29), siRNA and Lipofectin®, first the peptide and Lipofectin® are mixed together in Opti MEM cell culture medium and siRNA is mixed into this mixture Was added to form a siRNA / peptide / Lipofectin® composition.

  In the preparation of siRNA / peptide / cationic lipid composition using any order of siRNA / peptide / cationic lipid composition using GRKKRRRQRRPPQGRKKRRQRRPPQGRKKRRQRRRRPPQ (SEQ ID NO: 30) or GEQIAQLIAGYIDIILKKKSK (SEQ ID NO: 31) It doesn't matter if you make.

  To make siRNA / melittin / Lipofectin®, siRNA and melittin were first mixed together in Opti MEM cell culture medium, and then Lipofectin® was added to this mixture.

  To make a siRNA / histone H1 / Lipofectin® composition, first add Histone H1 and Lipofectin® together to the OptiMEM cell culture medium and mix well, then add siRNA. And fully mixed with the histone-Lipofectin® mixture to form a siRNA / histone H1 / Lipofectin® composition.

Table 3:

  Based on the previous results, while a representative polynucleotide delivery-enhancing polypeptide of the present invention can significantly enhance the uptake of siRNA into cells, the specific siRNA / polynucleotide delivery-enhancing polypeptide mixture of the present invention It is clear that the addition of any cationic lipid can substantially improve siRNA delivery efficiency.

Example 2
Production and Characterization of Compositions Comprising siRNA Coupled to a TAT-HA Polynucleotide Delivery-Promoting Polypeptide In this example, synthesis and uptake activity of a specific peptide covalently linked to one strand of a siRNA duplex Is described. These conjugates efficiently deliver siRNA into the cytoplasm.

Peptide synthesis peptides were synthesized by solid-phase Fmoc chemistry on a CLEAR-amide resin using a Rainin Symphony synthesizer. The coupling step was carried out over 40 minutes using 5 equivalents of HCTU and Fmoc amino acid with an excess of N-methylmorpholine. Fmoc detachment was achieved by treating the peptide resin twice with 20% piperidine in DMF with a period of 10 minutes. After completion of the entire peptide, the Fmoc group was removed using piperidine and washed thoroughly with DMF. Maleimide modified peptides were 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 degree of coupling was monitored by the Kaiser test. The peptide was cleaved from the resin by the addition of 10 mL of TFA with 2.5% water and 2.5 triisopropylsilane, and then gently stirred for 2 hours at room temperature. The resulting crude peptide was recovered by trituration with ether followed by filtration. The crude product was dissolved in Millipore water and lyophilized. The crude peptide was dissolved in 15 mL water and 3 mL acetic acid containing 0.05% TFA and reverse phase Zorbax RX-C8 (22 mm diameter) at a flow rate of 5 mL / min through a 5 mL injection loop. × 250 mm, particle size 5 μm). Purification was achieved by a linear AB gradient of 0.1% B / min where solvent A is 0.05% TFA in water and solvent B is 0.05% TFA in acetonitrile. The purified peptide was analyzed by HPLC and ESMS.

Both conjugate synthetic peptides and RNA are prepared by standard solid phase synthesis methods. Peptides and RNA molecules must be functionalized with special moieties in order to allow covalent bonding between them. For peptides, for example, the N-terminus is functionalized with 3-maleimidopropionic acid. However, it is recognized that other functional groups such as bromo or iodoacetyl moieties will also work. For RNA molecules, the 5 ′ end of the sense strand or the 3 ′ end of the antisense strand is functionalized with a 1-O-dimethoxytritylhexyl disulfide linker, for example, according to the following synthesis method.

With 0.393 mg (3 eq) of tris (2-carboxyethyl) phosphine (TCEP) in 0.3 ml of 0.1 M triethylamine acetate (TEAA) buffer (pH 7.0) over 3 hours at room temperature. A 5 ′ modified C6SS oligonucleotide (GCAAGCUGACCCUGAAGUUCAU (SEQ ID NO: 34); 3.467 mg; 0.4582 μmol) was reduced to the free thiol group. Reduced oligonucleotides in less than 20 minutes, Xterra (XTerra) (registration using 0-30% _CH 3 CN linear gradient in 0.1 M TEAA buffer (pH 7) to _(t r = 5.931 min) Purified by RP HPLC on a MS C ₁₈ 4.6 × 50 mm column.

Purified reducing oligonucleotide (1.361 mg, 0.19085 μmol) was dissolved in 0.2 ml of 0.1 M TEAA buffer (pH = 7), and then a peptide (0.79 mg, 0.79 mg, 1.5 equivalents) was added to the oligonucleotide solution. A precipitate formed immediately after addition of the peptide but disappeared when 150 μl of 75% CH ₃ CN / 0.1 M TEAA was added. After stirring overnight at room temperature, the resulting conjugate was purified within 20 minutes with a 0-30% CH ₃ CN linear gradient in 0.1 M TEAA buffer (pH 7) followed by a 5-minute period. Purification by RP HPLC on an XTerra® MS C ₁₈ 4.6 × 50 mm column with 100% C (t _r = 21.007 min). The amount of conjugate was measured spectrophotometrically based on the molar extinction coefficient calculated at λ = 260 nm. MALDI mass spectrometry showed that the peak observed for the conjugate (10 585.3 amu) was consistent with the calculated mass. Yield: 0.509 mg, 0.04815 μmol, 25.2%.

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

Structure of peptide-siRNA conjugate (SEQ ID NOs: 34 and 35)

Cells were seeded the day before in a 24-well plate so that the cells were ~ 50-80% confluent at the time of the uptake experimental transfection. For the complex, siRNA and peptide were diluted in OptiMEM® medium (Invitrogen) and then mixed for 5-10 minutes prior to addition to cells washed with PBS to complex. At each peptide concentration (2-50 μM), the final siRNA concentration was 50 OnM. The conjugate, also diluted in OptiMEM® medium, was added to the cells at final concentrations ranging from 62.5 nM to 500 nM. At a concentration of 50 OnM, it was also mixed with 20% FBS just prior to addition to the washed cells. Cells were transfected for 3 hours at 37 ° C., 5% CO ₂ . Cells were washed with PBS, treated with trypsin and then analyzed by flow cytometry. SiRNA uptake was measured by the fluorescence intensity of Cy5, and cell viability was evaluated by addition of propidium iodide.

  As shown in FIG. 1, peptide / siRNA conjugates achieve a higher percent uptake in peptide tail fibroblasts than peptide / siRNA complexes. Furthermore, the peptide / siRNA conjugate exhibits a higher mean fluorescence intensity (MFI; FIG. 2) than the peptide / siRNA complex. Thus, these data indicate that in certain embodiments, it is desirable to attach a polynucleotide delivery-enhancing polypeptide to the siRNA molecule.

Example 3
This example demonstrates that the screening of siRNA / delivery peptide complexes demonstrates the effective induction of siRNA uptake in 9L / LacZ cells by a variety of rationally designed polynucleotide delivery-enhancing polypeptides. Further evidence is provided that a wide variety of polynucleotide delivery-enhancing polypeptides of the present invention designed to promote siRNA uptake when complexed with siRNA.

Approximately 10,000 9L / lacZ cells per well were seeded in flat-bottomed 96-well plates so that they were -50% confluent at the next day's transfection. FAM-labeled siRNA and peptide were diluted in OptiMEM® medium (Invitrogen) to a final concentration of 1/2. Equal amounts of siRNA and peptide were mixed, complexed at room temperature for 5-10 minutes, and 50 μL was added to cells previously washed with PBS. Cells were transfected for 3 hours at 37 ° C., 5% CO ₂ . Cells were washed with PBS, treated with trypsin and then analyzed by flow cytometry. SiRNA uptake was measured by the intensity of FAM fluorescence and cell viability was assessed by the addition of propidium iodide. Table 4 below summarizes the percent cellular uptake data in 9L / LacZ cells for various rationally designed polynucleotide delivery-enhancing polypeptides. Table 4 also includes the peptide and siRNA concentrations used.

Table 4:

Example 4
In this example, siRNA delivery is facilitated by a polynucleotide delivery-enhancing polypeptide , and in this example, the polynucleotide delivery-enhancing polypeptide of the invention promotes siRNA uptake in LacZ cells, mouse primary fibroblasts and human monocytes. Is illustrated. The materials and methods used in the experiments performed with 9L / LacZ cells and mouse fibroblasts are generally the same as for mouse experiments, except that 9L / LacZ cells are replaced by mouse tail fibroblasts (MTF). The same as above. Materials and methods used in experiments performed with human monocytes will be described later. The results of transfection performed using MTF cells are summarized in Table 5. Table 5 also includes the amino acid sequence of the peptide used and the concentration of Cy5 label bound to the peptide and eGFP siRNA. The results of transfection performed using both MTF and 9L / LacZ cells are summarized in Table 6. The data shown in Table 6 provides a comparison of transfection efficiency for multiple peptide / siRNA complexes in different cell types.

Table 5:

Table 6:

  To further characterize the ability of polynucleotide delivery-enhancing polypeptides to transfect cells in culture, human monocytes are combined with 200 nM FITC-labeled siRNA complexed with various concentrations of PN73, PN250, PN182, PN58 and PN158. Incubated. Since human monocytes are the target cell type in the treatment of rheumatoid arthritis, human monocytes were used in addition to LacZ cells and mouse fibroblasts.

  Fresh human blood samples from healthy donors were purchased from Golden West Biologicals. To isolate monocytes, immediately after receipt, blood samples were diluted 1: 1 with PBS. Peripheral blood mononuclear cells (PBMC) were first isolated from whole blood by a Ficoll (Amersham) gradient. Monocytes were further purified from PBMCs using Miltenyi CD14 positive selection kit and supplied protocol (MILTENYI BIOTEC). To assess the purity of monocyte samples, cells were incubated with anti-CD14 antibody (BD Biosciences) and then sorted by flow cytometry. The purity of the monocyte sample was over 95%.

The following description is a brief overview of the transfection protocol used in this example. For adherent cell lines, cells were seeded at a confluency of 70-90%, and for suspended cells, 100,000 cells were seeded in each well. For the siRNA / transfection reagent complex, Cy5- or FAM-conjugated siRNA and peptide were diluted separately in OptiMEM® medium to a final concentration of 1/2. Equal amounts of siRNA and transfection reagent were mixed and allowed to complex for 5-10 minutes at room temperature. For siRNA-peptide conjugates, the conjugates were diluted directly in OptiMEM® medium. The transfection mixture was added to cells previously washed with PBS. Cells were transfected for 3 hours at 37 ° C., 5% CO ₂ . For analysis of siRNA uptake, cells were washed with PBS, treated with trypsin (adherent cells only) and then analyzed by flow cytometry. SiRNA uptake was measured by the intensity of intracellular Cy5 or FAM fluorescence. The cell viability was measured by propidium iodide (uptake) or annexin V-PE (staining).

  Previous experiments have shown that PN73, a representative polynucleotide delivery-enhancing polypeptide, is an ideal candidate for the treatment of rheumatoid arthritis. FIG. 3 illustrates the ability of several different polynucleotide delivery-enhancing polypeptides to promote siRNA uptake in human monocytes in culture. Lipofectamine transfection was used for comparison. Cell viability was also assessed for each peptide (Figure 4). These data are surprising that the PN73 peptide has been transfected into human monocytes with high efficiency and low toxicity, indicating that the peptide is an ideal candidate for the treatment of rheumatoid arthritis in vivo. An unexpected discovery is shown.

Example 5
siRNA delivery is facilitated by binding siRNA to a polynucleotide delivery-enhancing polypeptide. In this example, siRNA uptake is induced or enhanced in 9L / LacZ cell lines and primary fibroblasts from the mouse tail. To provide the results of a screen that evaluates the activity of the siRNA / polynucleotide delivery-enhancing polypeptide conjugates. The materials and methods used in these tests are generally the same as described above, except that the siRNA / peptide mixture required for the preparation of the siRNA / peptide complex is not required. The percent uptake for transfection performed with 9L / LacZ cells is summarized in Table 7. Table 7 also includes the peptides used and the peptide / siRNA conjugate concentrations. FAM-β-gal label bound to siRNA molecules was used. The results for transfection performed with MTF are summarized in Table 8. Table 8 also contains the peptide / siRNA conjugate used and the concentration of Cy5 label bound to the eGFP siRNA molecule.

Table 7:

Table 8:

  The previous data demonstrates that the various siRNA / peptide conjugates of the present invention mediate siRNA delivery to different cell types with high efficiency.

Example 6
In this example, knockdown of gene expression by siRNA is facilitated by a polynucleotide delivery-enhancing polypeptide complexed to siRNA. In this example, the efficiency of expression of the target gene by the siRNA / polynucleotide delivery-enhancing polypeptide complex of the present invention. A successful knockdown. In this study, the expression of the human tumor necrosis factor-alpha (hTNF-α) gene, which is thought to be associated with mediating the onset or progression of RA when overexpressed in human and other mammalian subjects, is a peptide The ability of the / siRNA complex to modulate was tested.

  Healthy human blood is purchased from Golden West Biologicals, California, from which peripheral blood mononuclear cells (PBMCs), Ficoll-Pague plus (Amersham) gradient Purified by Human monocytes were then purified from the PBMC fraction using MILTENYI BIOTEC magnetic microbeads. Isolated human monocytes were resuspended in IMDM supplemented with 4 mM glutamine, 10% FBS, 1 × non-essential amino acids and 1 × penicillin-streptomycin and stored at 4 ° C. until use.

  Human monocytes were seeded in a flat-bottomed 96-well plate in OptiMEM medium (Invitrogen) at 100,000 per 100 μl per well. Transfection reagents and siRNA were mixed in OptiMEM medium at the desired concentration for 20 minutes at room temperature (for Lipofectamine 2000 (Invitrogen)) or 5 minutes (for peptides). At the end of the incubation, FBS was added to the mixture (final concentration 3%) and 50 μl of the mixture was added to the cells. Cells were incubated for 3 hours at 37 ° C. Following transfection, the cells were transferred to a V-bottom plate and pelleted at 1500 rpm for 5 minutes. Cells were resuspended in growth media (IMDM with glutamine, non-essential amino acids, and penicillin-streptomycin). After overnight incubation, cells were stimulated with 1 ng / ml LPS (Sigma) for 3 hours. Following incubation, cells were harvested as described above for mRNA quantification and the supernatant was saved for protein quantification.

  For the measurement of mRNA, branch DNA technology from Genospectra (California) was used according to the manufacturer's instructions. In order to quantify the intracellular mRNA concentration, the mRNA of both the housekeeping gene (cypB) and the target gene (TNF-α) is measured, and the measured value for TNF-α is normalized to cypB to obtain relative luminescence. A unit (relative luminescence unit) was obtained. To quantify the protein concentration, a BD Bioscience TNF-α ELISA was used according to the manufacturer's instructions.

  As illustrated in Table 9 below, siRNAs were directed to target different regions of TNF-α mRNA. For each oligo listed in Table 9, no 3 'overhang (eg, dNdN where N represents any nucleotide) is not displayed

Table 9:

  Tables 10, 11 and 12 illustrate the efficiency with which specific oligos conjugated to the polynucleotide delivery-enhancing polypeptides of the invention are knocked down targeting TNF-α gene expression levels in human monocytes. ing.

Table 10:

Table 11:

Table 12:

  According to the previous results (Tables 10, 11 and 12), by using the novel siRNA / polynucleotide delivery-enhancing polypeptide composition of the present invention, an effective level of TNF-α gene expression knockdown in mammalian cells. Proved to be achievable.

Screening and Characterization In human monocytes (CD14 +) treated with LPS, TNF-α specific mRNA is induced within about 2 hours, followed by a peak concentration of TNF-α protein 2 hours later. SiRNA candidates are screened for knockdown activity by transfecting siRNA candidate sequences into monocytes using Lipofectamine 2000, treating infected cells with LPS, and measuring TNF-α mRNA levels after approximately 16 hours. did. 56 siRNA sequences were designed and screened for the ability to knock down TNF-α mRNA and protein concentrations in primary human monocytes in which these siRNAs were activated. The activity of a representative set of 27 siRNA sequences varied from 80% mRNA knockdown activity to a range of undetectable activities. In general, the TNF-α protein concentration is lower than the mRNA concentration, for example, 50% knockdown in TNF-α mRNA (TNF-α-1) is 75% at the TNF-α protein concentration. Caused a decrease in. A dose-response curve of the selected siRNA was obtained that showed a knockdown level of 30% to 60%. The calculated IC ₅₀ value was in the range of 10 pMolar to 200 pMolar. The evaluated siRNA sequences were distributed throughout the TNF-α transcript, but the most potent siRNA identified was located in two regions: the middle of the coding region and the 3′-UTR It is.

Example 7
In this example, where knockdown of gene expression by siRNA is facilitated by a polynucleotide delivery-enhancing polypeptide conjugated to siRNA, knockdown of expression of the target gene by the peptide-siRNA conjugate of the invention is demonstrated. The materials and methods for these tests are similar to those described above, except that no siRNA and peptide mixing is required. In this series of tests, knockdown experiments include peptide / siRNA mediated knockdown comparisons with or without lipofectamine. The results of this example are illustrated in Table 13 below.

Table 13:

  Based on the previous data (Table 13), the various polynucleotide delivery-enhancing polypeptides of the invention conjugated to siRNA have the function of promoting siRNA-mediated knockdown of TNF-α gene expression in mammalian subjects. Is proved.

Example 8
Time course of siRNA gene expression knockdown This example provides a test for time course of siRNA-mediated gene expression knockdown. To test the duration of the siRNA effect, the siRNA transfection procedure described above was employed except that fibroblasts from eGFP expressing mice were used. The transfection reagent used in this example was Lipofectamine. Cells were replated on day 18 due to overgrowth. The second transfection was performed 19 days after the first transfection. On day 19, eGFP concentration was measured after transfection. Along with GFPI siRNA (GFPI) and hairpin siRNA (D # 21), scrambled siRNA (scramble siRNA) or nonsense siRNA (nonsense siRNA) (Qiagen) was used as a control. Knockdown activity was normalized with scrambled siRNA (Qiagen control). Higher values indicate higher knockdown activity.

Table 14:

  Previous tests (Table 14) revealed siRNA knockdown activity around day 3 and lasted until day 9, after which the target gene expression was at baseline levels around day 17 It is shown that it recovered to. Decreased eGFP expression has occurred again after the second transfection on the 18th day, and the reagent can be repeatedly administered to cells to cause repetitive or sustained gene expression knockdown Is shown.

Example 9
Dose Dependence of TNF-α Gene Expression Knockdown Mediated by siRNA Complexed with Polynucleotide Delivery Accelerating Polypeptides This example is mediated by siRNA complexed with PN73, a representative polynucleotide delivery enhancing polypeptide. It is demonstrated that the knockdown activity in activated human monocytes is dependent on the administered concentration of the peptide-siRNA complex.

  The PN73 / siRNA complex was provided at a constant ratio between PN73 and siRNA of approximately PN73: siRNA = 82: 1 (Table 15). 400 nanomolar siRNA was complexed with 33 μM PN73 in OptiMEM medium for 5 minutes. After conjugation, the complex was serially diluted with OptiMEM (1: 2 ratio). The complex was added to human monocytes for transfection. According to the above, the following induction and mRNA quantification were performed.

Table 15:

  In a related series of experiments, siRNA was serially diluted and mixed with a fixed amount of PN73 (1.67 μM). In other words, the PN73 polynucleotide delivery-enhancing polypeptide was complexed with a titered amount of siRNA. PN73 (1.67 μM) was complexed with each titration of LC20 siRNA for 5 minutes at room temperature in OptiMEM medium. Following complexation, the complex was added to human monocytes for transfection. The induction and mRNA quantification data presented in Table 16 below was obtained by the above method.

Table 16:

Example 10
Multi-Dose Protocol for Prolonging Knockdown Effect by siRNA in Mammalian Cells This example demonstrates that in multi-dose regimes where the multi-dose regime is mediated by the siRNA / polynucleotide delivery-enhancing polypeptide composition of the invention. To effectively extend the gene expression knockdown effect of. The materials and methods used in these tests are the same as described above, except that transfections were repeated at the indicated times. Scrambled siRNA (Qiagen) was used as a parallel control. Data for multiple transfections using peptide / siRNA complexes are summarized in Table 17. The percent knockdown activity of the TNF-α gene represents the percent of total gene expression.

Table 17:

  The previous results (Table 17) show that when multiple transfections were performed in a timely manner (in the present example, about 5 to 7 days after the first transfection) It shows that the TNF-α gene expression knockdown effect can be maintained or re-induced.

Example 11
In vivo siRNA / peptide-mediated TNF-α gene expression knockdown activity In this example, the siRNA / polynucleotide delivery-enhancing polypeptide composition of the invention mediates systemic delivery and regulates expression of a target gene. Provides in vivo data demonstrating the efficacy of mediating therapeutic gene knockdown by siRNA effective to alter the phenotype of cells for therapeutic purposes.

  Human TNF-α expressing mice were purchased at 5 weeks of age from the Helenic Pasteur Institute, Greece. Mice were given intravenous (IV) route, 300 μl saline twice a week (4 mice), RA drug remicade (5 mg / kg) once a week (2 mice), or 1: LC20 siRNA (2 mg / kg) mixed with PN73 at a molar ratio of 5 was administered twice a week (2 mice). During the injection period, plasma samples were collected for the ELISA study (R & D Systems) and paw scores were taken twice a week as a generally accepted indicator for RA disease progression and treatment effects. . The plasma concentrations of TNF-α protein in the treated mice are shown in Table 18 below.

Table 18:

  The previous results demonstrate an effective reduction in TNF-α protein concentration in the circulating blood of peptide / siRNA treated mice compared to mice treated with remicade or saline (control). Further evidence of the in vivo effects of the siRNA / polynucleotide delivery-enhancing polypeptide compositions and methods of the invention is a generally accepted phenotypic indicator for the pathology and therapeutic effects of RA from the subject mice described above. Obtained using paw score.

Due to the difference in starting points (some animals present scores at earlier points), in this experiment the scores were set to zero for all animals. A score of 0 to 3 is presented for each foot, but the maximum score is 12 according to the following scoring index.
0: Normal 1: Foot or ankle joint edema or distortion 2: Foot or ankle joint distortion 3: Wrist or ankle joint stiffness

  The results of these foot score evaluations are illustrated in FIG. These data show that the polynucleotide delivery-enhancing polypeptide PN73, when injected into animals, shows a therapeutic amount of siRNA (eg, LC20, TNF-α2, and It demonstrates that TNF-α9 (UAGCCCAUGUGUGUAGCAAA (SEQ ID NO: 187)) can be delivered. Mice treated with PN73 / siRNA recovered better in paw scoring index at 8 weeks compared to mice treated with Remicade. When the paw score assessment was performed 11 weeks after treatment, the PN73 / LC20 complex achieved a paw score assessment comparable to that of the Remicade treated mice. In a 1: 5 ratio of PN73 peptide / LC20 siRNA, 2 mg / kg LC20 achieved the greatest delay in relative observations in RA progression compared to the lower dose LC20 tested. The relative effects of multiple siRNAs for the five different groups evaluated after treatment with PN73 and siRNA are summarized in Table 19 below.

Table 19:

The previous results show that the composition of siRNA and the polynucleotide delivery-enhancing polypeptide of the present invention provides a promising new therapeutic tool to regulate gene expression to treat and manage disease. . The siRNA of the present invention, such as siRNA targeting human TNF-α specific mRNA for degradation, has high specificity, low immunity compared to current treatment of RA with small molecules, soluble receptors, or antibodies. Produces virulence and high disease relief. More than 50 candidate siRNA sequences were screened targeting hTNF-α that produced 30-85% knockdown in a single dose. Comparing more than 20 computer-designed peptide conjugates and / or covalent molecules for uptake of fluorescent RNA by monocytes, many showed significantly better uptake than siRNA conjugated to lipofectamine or cholesterol, less than 10 pM Was found to have an IC ₅₀ value of The peptide-siRNA formulation effectively knocks down the concentration of TNF-α mRNA and protein in activated human monocytes in vitro.

  One representative candidate delivery peptide / siRNA formulation was evaluated in a transgenic mouse model of rheumatoid arthritis (RA) that constitutively expresses two types of human TNF-α. Animals treated with 2 mg / kg siRNA or infliximab by IV injection twice weekly from 6 weeks of age showed stabilization of the RA score (inflammation of the foot and joints) from 7 weeks of age, compared with this In some controls, these conditions persisted until week 10. At 9 weeks of age, animals treated with siRNA showed a similar decrease in RA score compared to animals treated with infliximab, but showed significantly lower plasma TNF-α protein concentrations.

  Based on the disclosure herein, the use of siRNAs that inhibit the expression of target genes such as cytokines such as TNF-α that play an important role in pathological conditions such as inflammation is exemplified in RA in mammalian subjects. An effective treatment is provided to alleviate or prevent symptoms of certain diseases. Alternative peptide / siRNA compositions for use within the methods and compositions of the invention are not by complexation with the product of the target gene, eg, TNF-α, as is the case with antibodies or soluble receptors. , Providing an advantage associated with the ability of the composition to reduce or eliminate target gene expression, such as TNF-α expression.

  Improved systemic delivery of nucleic acids according to the teachings of the present invention provides additional advantages for developing siRNA as drugs. Specific challenges in this context include delivery to the target cell or tissue through the tissue barrier, maintaining the stability of the siRNA, and delivering a sufficient amount of siRNA through the cell membrane into the cell. Intracellular delivery is included. The present disclosure targets specific gene expression, such as human TNF-α expression, that mitigates disease activity in transgenic model animals that are predictive of the target disease, as exemplified by studies with RA model mice. For the first time, an effective delivery system in vivo comprising the novel peptide / siRNA composition is shown. In a related study, the compositions and methods of the present invention effectively inhibit TNF-α expression in activated monocytes from RA patients. These results indicate that the RNAi pathway effectively mediates changes in cellular phenotype and disease progression through intracellular effects on the TNF pathway, and is further characterized by residual immunity characteristic of RA treatment with current antibodies. It is shown to avoid toxic effects resulting from the circulation of reactive antibody / TNF-α complexes. In particular, so that the siNA and polynucleotide delivery-enhancing polypeptide doses shown in these examples always correlate with levels of cell viability of at least 80-90% or above, The accompanying toxic effects were minimized.

Example 12
Optimization of rational design of polynucleotide delivery-enhancing polypeptides This example provides representative designs and data for optimizing polynucleotide delivery-enhancing polypeptides of the invention. The rational design manipulations of interest were performed on polynucleotide delivery-enhancing polypeptides derived from histone H2B.

Table 20:

  Table 20 provides an illustration of the primary structure of PN73 and PN73 derivatives made to optimize the rational design of PN73-based polynucleotide delivery-enhancing polypeptides. The gray colored C-terminus of each peptide represents the hydrophobic domain of the peptide, and the black N-terminus of each peptide represents the hydrophilic domain. The original peptide, PN73, is shown above as an example of a polynucleotide delivery-enhancing polypeptide for inducing or facilitating siRNA delivery to cells.

  To better understand the function-structure activity relationship between this polynucleotide delivery-enhancing polypeptide and other polynucleotide delivery-enhancing polypeptides, C-terminal and N-terminal functions and conjugates with PN73 and other chemical moieties Primary structure tests were performed by characterizing the activity of

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

PN73, PN360, and PN361 are peptide fragments of H2B, and the portion of the H2B protein to which they correspond is specified in parentheses after the following peptide names. The amino acid sequences of PN360 and PN361 listed below are aligned with the corresponding amino acid sequences found in PN73. The PN73 peptide fragment is underlined in the H2B amino acid sequence and corresponds to amino acids 13 to 48 of H2B. The PN73 peptide fragment may correspond to 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 PN73. A PN73 conjugate is a PN73 covalently linked to a single siRNA strand (eg, sense strand). PN404 is a version of PN73 in which all lysines are substituted with arginine, and PN509 is a PEGylated PN73 (PEG molecular weight 1 kDalton) derivative in which the N-terminus is PEGylated.

H2B (Histone 2B) amino acid sequence MPEPAKSAPAPK KGSKKAVTKKAQKKDSKKRKRRSKESYSVYVYKVLK VHPDTGISKAMGIMNSFVNDIFFERIAGEASRLAHYNKRSTGSKTLKTS

PN73 (13-48)
NH ₂ -KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ- amide (SEQ ID NO: 59)

PN360 (13-35; N-terminus 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) conjugate siRNA-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 59)

PN404 (PN73 with all lysines replaced with arginine)
NH ₂ -RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ- amide (SEQ ID NO: 91)

PN509 (PEGylated PN73)
PEG-RGSRRAVTRAQRRDGGRRRRRSRRESYSVYVYRVLRQ-amide (SEQ ID NO: 90)

  FIG. 6 presents the results of a test of uptake efficiency and survival in mouse tail fibroblasts for a polynucleotide delivery-enhancing polypeptide, a rationally designed derivative of PN73. Yes. The changes in the activity of modified PN73 in mouse tail fibroblasts are shown graphically. Unlike PN404, PN509 increases uptake without increasing toxicity. Deletion of part of the N-terminus of PN73 reduces activity, but removal of the C-terminal residue abolished activity. Both PN73 and PN509 show higher activity in primary cells than Lipofectamine (Invitrogen, California).

Example 13
The purpose of this example is that acetylated polynucleotide delivery-enhancing polypeptides exhibit high stability in plasma. The purpose of this example is that modification of a representative polynucleotide delivery-enhancing polypeptide PN73 provides improved stability to the peptides. The result was to determine if the transfection activity was promoted as a result. The plasma stability of unmodified PN73, N-terminal PEGylated PN73 and N-terminal acetylated PN73 was compared. The C-terminal of PN73 is amidated. Using size exclusion chromatography coupled to a UV detector, the stability of unmodified and modified PN73 was characterized before and after incubation in plasma.

  In the absence of plasma, unmodified, PEGylated and acetylated PN73 showed a different but overlapping UV trace at about 9 minutes. However, after exposure to plasma for 4 hours, there was no UV trajectory specific to unmodified PN73 and PEGylated PN73, suggesting significant degradation. In contrast, a unique UV trajectory remains for acetylated PN73, and this modification significantly increased the stability of PN73 in plasma compared to unmodified PEG73 and PEGylated PN73. Indicated.

These data indicate the surprising and unexpected discovery that the stability of PN73 in plasma can be improved by N-terminal acetylation of the PN73 peptide. The primary structure of the acetylated PN73 peptide is as follows:
Ac-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 59)

Example 14
Polynucleotide Delivery- Enhanced Polypeptide Does Not Induce Interferon Response The purpose of this example is to interferon cells transfected with either a liposome reagent and siRNA, or a representative polynucleotide delivery-enhancing polypeptide, PN73 peptide and siRNA It was to compare the reactions. Interferon reactivity was assessed by ELISA (protein) and bDNA (mRNA level).

  Traditionally, siRNA molecules are delivered into cells by liposome-mediated transfection. However, this generally results in low-efficiency delivery, resulting in an in vivo inflammatory response and, consequently, upregulation of interferon gene expression that inhibits cell proliferation. As a result, there is a limit to the reduction in target gene expression levels, thus making siRNA ineffective as a therapeutic and gene expression research tool. SiRNA delivery by PN73 overcomes this challenge.

  FIG. 7 shows the results of a bDNA assay of transfection of several different siRNAs with Lipofectamine versus PN73 peptide. siRNA was complexed with either lipofectamine or PN73 at concentrations of 1 nM, 10 nM, 100 nM or 200 nM. Interleukin 1β (IL-1β) functioned as a molecular marker for measuring interferon reactivity, and Qneg was used as a negative control. As shown in FIG. 7, lipofectamine complexed with 100 nM or 200 nM TNF-α9 siRNA resulted in a significant increase in IL-1β mRNA concentration. Furthermore, all other siRNAs tested also caused a gradual increase in IL-1β mRNA concentration. In contrast, the same siRNA complexed with the PN73 peptide did not cause an increase in IL-1β mRNA concentration.

  To further characterize the differences in interferon reactivity observed between cells transfected with either Lipofectamine and PN73 transfection, an ELISA assay was performed to measure protein expression levels of the following molecular markers: Leukin 1β (IL-1β), interferon α (INF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 12 (IL-12), MIP-1α, interferon γ ( IFN-γ), and tumor necrosis factor-α (TNF-α). Table 21 summarizes the relative protein expression levels of cells transfected with lipofectamine complexed with siRNA or PN73 complexed with siRNA.

Table 21:

  As shown in Table 21, both LC20 and LC17 siRNAs showed no interferon reaction regardless of which transfection reagent was used. However, transfection of IFN-1 or TNF-α9 with lipofectamine resulted in increased protein expression levels of IL-1β, IL-6, and MIP-1α. In contrast, transfection of siRNA with all PN73 tested did not observably induce protein expression of any interferon response marker tested.

  These data from the ELISA assay indicate a surprising and unexpected finding that transfection of siRNA mediated by PN73 does not induce an interferon response.

Example 15
The purpose of this example is that siRNA bound to a polynucleotide delivery-enhancing polypeptide exhibits a higher knockdown activity than siRNA complexed with the polynucleotide delivery-enhancing polypeptide. The purpose is to compare the knockdown activity of bound or complexed LC13 and LC20 siRNAs in human monocytes. The methods used for the isolation and transfection of human monocytes and the measurement of knockdown activity have been discussed previously. Qneg represents a random siRNA sequence and serves as a negative control in these experiments. The observed Qneg knockdown activity is normalized to 100% (100% gene expression level) and the activity of LC20 and LC13 is expressed as a relative percentage of the negative control. LC20 and LC13 are siRNAs that target the human TNF-α gene. FIG. 8 shows LC20 and LC13 siRNA without PN73 (FIG. 8-C), LC20 and LC13 siRNA complexed with PN73 (FIG. 8-B), or LC20 and LC13 siRNA conjugated with PN73 (FIG. 8- The knockdown activity of A) is shown. LC20 and LC13 were tested over a concentration range of 0 nM to 2.5 nM. PN73 was kept in a 1: 1 ratio in both the complex and conjugate experiments.

  As expected, in the absence of PN73, LC13 and LC20 showed little knockdown activity (FIG. 8-C). Both LC13 and LC20, when complexed with PN73, resulted in about 15% and 30% reduction in TNF-α gene expression compared to the Qneg control (FIG. 8-B). However, knockdown activity for TNF-α was reduced to less than 60% when siRNA bound to PN73 (FIG. 8-A). This is a significant increase in knockdown activity by siRNA compared to the PN73 / siRNA complex. Thus, these data represent a surprising and unexpected finding that siRNA knockdown activity is significantly improved when siRNA is bound to a representative polynucleotide delivery-enhancing polypeptide PN73.

Example 16
Inhibition of cholesterol-promoting siRNA uptake by serum is rescued by polynucleotide delivery-enhancing polypeptides In this example, the addition of a permeable peptide to a delivery formulation comprising siRNA bound to a cholesterol moiety results in cholesterol-siRNA It demonstrates that the inhibitory effect of sera on the uptake of is reduced in a concentration-dependent manner. For analysis of siRNA uptake, cells were washed with PBS, treated with trypsin (adherent cells only) and then analyzed by flow cytometry. Uptake of siRNA defined as BA was also measured by intracellular Cy5 or FAM fluorescence intensity as described above, and cell viability was assessed by the addition of propidium iodide or annexin V-PE. To distinguish cell uptake from fluorescently labeled siRNA insertion into the membrane, trypan blue was used to quench the fluorescence on the cell membrane surface.

Table 22:

  The data in Table 22 shows that the presence of serum significantly reduces the cellular uptake of siRNA bound to the cholesterol moiety according to the present invention. However, cellular uptake of unbound siRNA is rescued in the presence of a typical delivery-enhancing peptide, PN73.

  Comparison of the cellular uptake of a permeant peptide delivery enhancer, PN73 complexed with cholesterol according to the present invention (cholesterol siRNA + PN73) and unconjugated siRNA (siRNA + PN73) complexed with PN73 went. As shown in FIG. 9, equivalent cell uptake activity can be achieved at high concentrations of PN73, either with unbound siRNA or with siRNA bound to cholesterol. For uptake assays associated with these assays, mice in OptiMEM® medium (Invitrogen) supplemented with serum at a constant or various concentrations of siRNA conjugated to cholesterol and siRNA / PN73 complexes. In the tail fibroblasts (MTF). For both cholesterol and complex, the final concentration of siRNA was 0.2 μM. Uptake efficiency and average fluorescence intensity were evaluated by flow cytometry.

  The ability of PN73 and also PN250 to rescue serum inhibition of cellular uptake of siRNA bound to cholesterol was further characterized in mouse tail fibroblasts (MTF). FIG. 10 illustrates the effect of high concentrations of fetal bovine serum (FBS) on siRNA cell uptake. In the absence of either PN73 or PN250, cellular uptake of siRNA bound to cholesterol is greatly attenuated by as little as 5% FBS. However, siRNA uptake is rescued in the presence of either 40 μM PN73 or 80 μM PN250.

  Previous data (Table 22 and FIG. 10) shows that siRNA binding to cholesterol significantly promotes uptake of siRNA into cells. However, the uptake of siRNA bound to cholesterol can be greatly reduced or eliminated by the presence of serum. This is probably due to the binding of the cholesterol moiety to serum proteins that inhibit the ability of cholesterol-binding siRNA to enter target cells. However, in the presence of delivery enhancers exemplified by the permeable peptides PN73 and PN250, inhibition of cell uptake by serum can be rescued. More specifically, the addition of a permeable peptide to a delivery formulation comprising siRNA linked to a cholesterol moiety reduces the inhibitory effect of serum on cholesterol-siRNA uptake in a concentration-dependent manner.

Example 17
Deletion analysis of representative polynucleotide delivery-enhancing polypeptides In this example, experiments used to optimize cellular uptake of siRNA and siRNA-mediated target gene knockdown activity by the representative polynucleotide delivery-enhancing polypeptide PN73 The design of is illustrated.

  Table 23 below shows a representative polynucleotide delivery-enhancing polypeptide PN73 and its truncated derivatives. The amino acid sequences of PN360 and PN361 listed below are aligned with the corresponding amino acid sequences of PN73. 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 PN73. PN766 corresponds to 15 C-terminal amino acids of PN73. PN73, PN360, PN361, and PN766 are not tagged on the C-terminal side with FITC (fluorescein-5-isothiocyanate) (that is, -GK [epsilon] G-amide). Furthermore, Table 23 shows 11 types of cleavages of a representative polynucleotide delivery-enhancing polypeptide PN73 created by stepwise deletion of 3 residues at a time from the N-terminus of the peptide except for PN768. The type is shown. All these peptides are tagged on the C-terminal side with FITC (fluorescein-5-isothiocyanate) label (ie, -GK [epsilon] G-amide). Can be detected and / or sorted by flow cytometry. It should be noted that PN766 and PN708 have the same amino acid sequence but differ in that PN708 has a FITC tag at the C-terminus. The following is a description of the primary structure of PN73 and the truncated form, which will be further tested for transfection activity.

Table 23:

Example 18
Deletion analysis of representative polynucleotide delivery-enhancing polypeptides The functional domain of representative polynucleotide delivery-enhancing polypeptide PN73 is critical for the ability of the polynucleotide delivery-enhancing polypeptide to efficiently deliver siRNA into cells. It is a thing. These functional domains include a membrane binding region, a fusion region and a nucleotide binding region. Briefly, membrane binding refers to the ability of a representative polynucleotide delivery-enhancing polypeptide to bind to the cell membrane. The fusion property represents the ability to detach from the cell membrane and enter the cytoplasm. The membrane-bound and fusion domains of the peptide are mechanistically close (ie, the ability of the peptide to enter the cell) and therefore can be difficult to distinguish experimentally. Therefore, these two characteristics (peptide domains) will be merged into a single analysis. Finally, nucleotide binding represents the ability of a peptide to bind to a nucleotide. The method used to determine each of the above-described domains of a polynucleotide delivery-enhancing polypeptide is discussed in more detail below. Data for determining the membrane binding / fusion domain and nucleotide binding domain of representative polynucleotide delivery-enhancing polypeptide PN73 are summarized in Table 24 (data for all concentrations tested are not shown).

Table 24:

Representative polynucleotide delivery-enhancing polypeptide membrane binding and cell uptake assays using fusion domain primary mouse tail fibroblasts (MTF) showed that the full-length or truncated form of the representative polynucleotide delivery-enhancing polypeptide PN73 The ability to enter cells was tested in vitro. The number of cells in culture receiving the FITC-labeled peptide was determined by flow cytometry. The percentage of cellular uptake of the peptide was expressed relative to the total number of cells present in the culture. In addition, the average fluorescence intensity (MFI) was used to evaluate the amount of FITC-labeled peptide found in the cells. MFI has a positive correlation with the amount of FITC-labeled peptide in the cell: high relative MFI values correlate with more amount of intracellular FITC-labeled peptide. The peptides in Table 23 were judged to be at concentrations of 0.63 μM, 2.5 μM and 10 μM; PN768 was tested at 2 μM, 10 μM and 50 μM.

  The day before transfection, cells were exposed to the full-length or truncated forms of the representative polynucleotide delivery-enhancing polypeptide PN73. FITC-tagged peptides were diluted in OptiMEM® medium (Invitrogen) for about 5 minutes at room temperature and then added to the cells. Cells were transfected for 3 hours and washed with PBS, treated with trypsin and then analyzed by flow cytometry. Cell viability was measured as described above. Cell uptake was distinguished from membrane insertion by using Trypan Blue to quench any fluorescence on the cell membrane surface.

  In the cell uptake assay, full length FITC-labeled PN73 peptide (PN690) achieved almost 100% cell uptake at all concentrations tested (10 μM in the column titled “% Peptide Cell Uptake” in Table 24). Results are shown). The other truncated forms of PN73, except for PN768, which required 50 μM, achieved the same percentage of cellular uptake (number in parentheses) as PN690 at a concentration of 10 μM. It was suggested that it is not required for its ability to enter cells. The five C-terminal residues of the representative polynucleotide delivery-enhancing polypeptide PN73, identified as PN768, were sufficient for cellular uptake of the peptide. However, although not shown in Table 24, it should be noted that the truncated form of PN73 showed a decrease in cellular uptake activity proportional to peptide length at 0.63 μM. In other words, the general observation for peptides tested at a concentration of 0.63 μM is that as the length of the PN73 peptide decreases, its cellular uptake activity decreases, and thus the cellular uptake activity of the peptide. Is suggested to be dose dependent.

  These results indicated that all truncated forms of representative polynucleotide delivery-enhancing polypeptides retain the ability to enter a high percentage of cells in culture.

  The cellular uptake assay showed that the truncated form of the representative polynucleotide delivery-enhancing polypeptide PN73 has similar cellular uptake activity, but MFI measurements indicated that the truncated form of the PN73 peptide was in the FITC that entered the cell. It was shown that discrimination was possible based on the average amount of labeled peptide. As shown in the column titled “Peptide FITC MIF” in Table 24, the full-length FITC-labeled PN73 peptide (PN690) showed a dose-dependent increase in MFI, but at 10 μM, The highest MFI of 125 units is achieved. PN661, PN685 and PN660 showed MFI levels comparable to full length PN73 (PN690). However, PN735 and PN655 showed low MFI levels of about 80 MFI units and 60 MFI units, respectively. In addition, a significant decrease in MFI detectability was observed with PN654, PN708, PN653, PN652, PN651, and PN768, and deletion of the 18 N-terminal residues of PN73 resulted in efficient uptake of the peptide by the cell. Suggested to be removed.

  These results show that all truncated forms of PN73 are able to enter cells in culture at a high percentage, and in particular, the first 18 N-terminal residues of the representative polynucleotide delivery-enhancing polypeptide PN73 are: It is essential for efficient uptake of peptides by cells in culture. These results also indicate that truncated derivatives of the representative polynucleotide delivery-enhancing polypeptide PN73 ((PN661; PN685; PN660; PN735; and PN655) can efficiently enter cultured cells.

  In summary, peptide cellular uptake data indicate that the C-terminal five residues of a representative polynucleotide delivery-enhancing polypeptide PN73 are sufficient for entry into the cell, This suggests that the membrane-bound / fusion domain of the peptide is located at the C-terminus. However, the FITC MFI data for the peptide shows that removal of the N-terminal 18 residues of the representative polynucleotide delivery-enhancing polypeptide PN73 limits the efficiency of the peptide entering the cell. , Suggesting that the N-terminus of the peptide is required for effective uptake properties of the membrane binding / fusion domain.

Nucleotide binding domains of representative polynucleotide delivery-enhancing polypeptides:
For each peptide with the series of deletions, the ability to complex with siRNA and deliver siRNA to primary MTF cells was measured by cell uptake assay and MFI. The nucleotide binding domain of a representative polynucleotide delivery-enhancing polypeptide PN73 was characterized by comparing the relative amount of cellular uptake of siRNA by various peptides having the series of deletions. All peptides showed a high percentage (> 40%) of siRNA cell uptake correlated with the presence of the nucleotide binding region. A low percentage of siRNA cell uptake (less than 30%) represents the absence of a nucleotide binding domain.

  To test the full-length and truncated forms of the representative polynucleotide delivery-enhancing polypeptide PN73, MTF cells were treated with siRNA and peptides conjugated to Cy5 or FAM as described above. After washing, the cells were treated with trypsin and then analyzed by flow cytometry. Intracellular uptake of siRNA was measured by intracellular Cy5 fluorescence intensity; trypan blue was used to quench any fluorescence on the cell membrane surface. Uptake was expressed relative to the total number of cells.

  Table 24 shows the resulting percentage of cellular uptake of siRNA by the peptide when using 0.5 μM Cy5-binding siRNA. Non-FITC labeled PN73 (PN643) peptide achieved about 100% siRNA incorporation at a concentration of 10 μM (data not shown). However, when the PN73 peptide is labeled with a FITC tag (PN690), the maximum cell uptake activity observed at 2.5 μM is reduced to about 60%, and the addition of the FITC label results in the cell uptake function of the peptide. Is suggested to be subject to interference. Thus, the cellular uptake activity of each peptide observed in this assay may not reflect the true siRNA cell uptake activity of the peptide. Nevertheless, as represented by PN661, a slight decrease in siRNA cell uptake activity was observed by removal of the three most N-terminal residues of the PN73 (PN690) peptide. Similarly, both PN660 and PN708 showed a modest decrease in siRNA cell uptake activity compared to full-length PN73 (PN690).

  These data indicate that the polynucleotide delivery-enhancing polypeptides of the present invention, including N661, PN660 and PN708, bind to nucleic acids. In contrast, a decrease in siRNA uptake activity was observed with PN685, PN735, PN655 and PN654. In PN653, PN652 or PN651, no significant siRNA uptake activity was observed, and the 12-amino acids at the C-terminus of the representative polynucleotide delivery-enhancing polypeptide (the 37th to 48th residues of the H2B protein) had a nucleotide binding It is suggested that the domain is not included. Furthermore, since the peptides PN661, PN660 and PN708 do not contain the 3-residue deletion of the full-length peptide (PN690), it is presumed that they retain siRNA binding activity. These data suggest that the ability to bind to the nucleic acid of the nucleotide binding domain present at the N-terminus of a representative polynucleotide delivery-enhancing polypeptide is sensitive to the presence of certain residues in the N-terminus. ing.

  In general, siRNA cell uptake activity of the PN73 deletion series indicates that PN708 (residues 34-48) corresponds to the smallest C-terminal fragment of the PN73 peptide required for siRNA cell uptake activity, This is shown by the data. This is consistent with the nucleotide binding domain of the representative polynucleotide delivery-enhancing polypeptide PN73 being located within the first 24 N-terminal residues.

  Regarding the ability to transfect cells with siRNA of the PN73 peptide deletion series, the PN73 peptide deletion series was further characterized by measuring the relative average amount of Cy5-binding siRNA that entered the cells by MFI. Delivery of Cy5-binding siRNA by full-length FITC-labeled PN73 peptide (PN690) achieved an MFI of approximately 50 relative units. The PN73 deletion series of peptides PN735, PN655, PN654 and PN708 showed low MFI in the range of about 34 to 44 units. The PN73 deletion series PN661, PN685 and PN660 showed MFI levels slightly above the full-length PN73 (PN690) peptide. In contrast, PN654, PN653, PN652, and PN651 show relatively little or no MFI (3 to 14 units), and little or no degree of Cy5-binding siRNA after transfection with these peptides. It indicates that the cell did not enter. The low MFI values observed for PN653, PN652 and PN651 indicate that the siRNA cell uptake data showed that PN654, PN653, PN652 and PN651 did not complex with siRNA or promote siRNA entry into cells. Was correlated.

  Using fluorescence microscopy images, the cellular localization of Cy5-labeled siRNA complexed with polynucleotide delivery-enhancing polypeptide PN73 was compared to the cellular localization of siRNA transfected with Lipofectamine (Invitrogen). The localization of siRNA delivered by Lipofectamine is characterized by more punctate staining, suggesting potential endosomal localization, whereas in the case of PN73, a more uniform perinuclear periphery Staining is shown. SiRNAs localized in endosomes cannot access the RISC complex in the cytoplasm and cannot silence the expression of the target gene. In contrast, the uniform cytoplasmic distribution of siRNA observed in the case of PN73-mediated delivery is essential for access to the RISC complex and decreased target gene expression. These results indicate that the polynucleotide delivery-enhancing polypeptides of the present invention sufficiently improve siRNA delivery and sufficiently improve target gene silencing (knockdown) compared to cationic lipids. .

  These data indicate that the ability of a representative polynucleotide delivery-enhancing polypeptide PN73 to promote highly efficient delivery of siRNA into cells depends on the 24 most N-terminal residues of the peptide. Show. These data also show that shortened derivatives of the representative polynucleotide delivery-enhancing polypeptide PN73 (PN661; PN685 PN660; PN735; PN655; and PN708) are efficiently complexed with siRNA into the siRNA. It can be delivered.

Analysis of truncated PN360 and PN361 polynucleotide delivery enhancing polypeptides:
Characterization of PN360 (C-terminal) and PN361 (N-terminal) in the siRNA cell uptake assay performed as described above demonstrates the relationship between the function-structure activity of the C-terminal region and N-terminal region of PN73. It was.

  Table 24 shows that the N-terminal deletion of PN73 (see PN361) reduced siRNA cellular uptake activity by 50%; removal of the C-terminal residue (see PN360) completely abolished siRNA cell uptake activity. It has been shown. These data indicate that the C-terminal domain of the representative polynucleotide delivery-enhancing polypeptide PN73 is required for the cellular uptake activity of nucleotides by the peptide.

Peptide-siRNA conjugates that facilitate delivery of covalently bound cargo into cells:
Peptide uptake activity and MFI measurements of truncated forms of representative polynucleotide delivery-enhancing polypeptides suggest that these peptides may serve as delivery vehicles for various molecular cargoes. Yes. This includes covalently linking the desired effector molecule comprising nucleic acid and peptide to full length PN73 or a derivative thereof. Limited delivery of effector molecules to specific cell types and / or intracellular organelles can be achieved by modifying delivery peptides with specific moieties (lipid, peptide and / or sugar groups). Will.

  Deletion analysis of representative polynucleotide delivery-enhancing polypeptides suggests that the N-terminus is critical for the ability of peptides to bind to nucleotides (eg, siNA) and deliver into cells. The However, even in the absence of the N-terminal residue, these non-nucleotide binding peptides and significantly weakened nucleotide binding peptides retain their membrane-bound and fusion domains (eg, PN361; PN735; PN655; PN654; PN653; PN652 and PN651, and their derivatives). Despite the fact that peptides cannot bind to nucleotides, these peptides are still used for siNA delivery into cells by covalently attaching siRNA to the peptide's membrane-bound and / or fusion domains. Is possible. Thus, the covalent binding of siRNA rescues the inability of the peptide to bind to the nucleotide and allows for effective peptide-mediated delivery of the siRNA into the cell. Furthermore, the siNA / peptide conjugate need not be limited to selection of truncated forms of representative polynucleotide delivery-enhancing polypeptides, and may comprise full-length PN73 and derivatives thereof.

  The following are non-limiting examples of methods used to create siNA / peptide covalent bonds. Both peptides and RNA molecules must be functionalized with special moieties in order to be able to covalently bind each other. For peptides, for example, the N-terminus is functionalized with 3-maleimidopropionic acid or the like. However, it is recognized that other functional groups such as bromo or iodoacetyl moieties will function as well. For RNA molecules, the 5 'end of the sense strand or the 3' end of the antisense strand is functionalized with a 1-O-dimethoxytritylhexyl disulfide linker, for example, according to the following synthetic method.

With 0.393 mg (3 eq) of tris (2-carboxyethyl) phosphine (TCEP) in 0.3 ml of 0.1 M triethylamine acetate (TEAA) buffer (pH 7.0) over 3 hours at room temperature. The 5 ′ modified siNA is reduced to a free thiol group. Reduced oligonucleotides are XTerra (registered) using a 0-30% CH ₃ CN linear gradient (t _r = 5.931 min) in 0.1 M TEAA buffer (pH 7) in less than 20 minutes. Purify by RP HPLC on a MS C ₁₈ 4.6 × 50 mm column.

A conjugate as described above was prepared. Purified reduced siNA (1.361 mg, 0.19085 μmol) is dissolved in 0.2 ml of 0.1 M TEAA buffer (pH = 7), and then a peptide having a maleimide moiety is coupled to the N-terminus of the peptide. (0.79 mg, 1.5 eq) was added to the siNA solution. Following the addition of the peptide, the precipitate was dissolved by the addition of 150 μl of 75% CH ₃ CN / 0.1M TEAA. After stirring overnight at room temperature, the resulting conjugate was added to a 0-30% CH ₃ CN linear gradient in 0.1 M TEAA buffer (pH 7) in less than 20 minutes, followed by less than 5 minutes. _100% C (t r = 21.007 min) was used Xterra of (XTerra) was purified by RP HPLC of (r) _MS C 18 4.6 × 50mm column. The amount of conjugate was measured spectrophotometrically based on the molar extinction coefficient calculated at λ = 260 nm. MALDI mass spectrometry shows that the peak (10585.3 amu) observed in the conjugate is consistent with the calculated mass.

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

  The results of cell uptake activity are shown in Table 25 below.

Table 25:

  The results in Table 25 indicate that the PN651-siRNA conjugate promotes siRNA uptake into cells. Next, MFI was measured. The results are shown in Table 26.

Table 26:

  The MFI results shown in Table 26 are consistent with the percent siRNA incorporation data shown in Table 25. These results indicate that the PN651-siRNA conjugate promoted siRNA uptake into cells.

Selected truncated gene target knockdown activities of representative polynucleotide delivery-enhancing polypeptides:
We have demonstrated effective knockdown of target gene expression by the siRNA / polynucleotide delivery-enhancing polypeptide complex of the present invention. In particular, the ability of siRNA / polynucleotide delivery-enhancing polypeptide complexes to modulate the expression of the human tumor necrosis factor-α (hTNF-α) gene was evaluated. The implication of targeting the hTNF-α gene is that it is involved in mediating the onset or progression of rheumatoid arthritis (RA) when the hTNF-α gene is overexpressed in humans and other mammalian subjects. It is in thought.

  Human monocytes were used as a model system to measure the effect of siRNA / polynucleotide delivery-enhancing polypeptide complexes on hTNF-α gene expression. Qneg represents a random siRNA sequence and served as a negative control. The observed Qneg knockdown activity is normalized to 100% (100% gene expression level), but the following knockdown activity of each A19S21, 21/21 and LC20 siRNA is expressed as a relative percentage of the negative control. did. A19S21, 21/21 and LC20 are siRNAs that target hTNF-α mRNA. Representative polynucleotide delivery-enhancing polypeptides PN643 (full-length PN73 with C-terminal label removed), PN690 (full-length PN73 with FITC tag at C-terminal), and cleavage of PN73 included in the deletion series PN660, PN735, PN654 and PN708 are complexed with A19S21, 21/21 and LC20 siRNAs, and the effect of these complexes on the ability of each siRNA to reduce hTNF-α gene expression levels in human monocytes. It was measured.

  The experiment was performed as follows: Human monocytes were seeded at 100 K / well / 100 μl in OptiMEM medium (Invitrogen) in flat-bottom 96-well plates. A representative polynucleotide delivery-enhancing polypeptide was mixed with 20 nM siRNA at a molar ratio of 1 to 5 at room temperature for 5 minutes in OptiMEM medium. At the end of the incubation, FBS was added to the mixture (final concentration 3%) and 50 μl of the mixture was added to the cells. Cells were incubated for 3 hours at 37 ° C. After incubation, the cells were transferred to a V-bottom plate and the cells were pelleted at 1500 rpm for 5 minutes. Cells were resuspended in growth media (IMDM with glutamine, non-essential amino acids, and penicillin-streptomycin). After overnight incubation, monocytes were stimulated for 3 hours to increase TNF-α expression levels by application of 1 ng / ml LPS (Sigma). Following induction with LPS, cells were harvested as described above for mRNA quantification and the supernatant was saved for cases where protein quantification was desired.

  For mRNA measurement, Genospectra's branch DNA technology was used according to the manufacturer's instructions. In order to quantify the intracellular mRNA concentration, the mRNA of both the housekeeping gene (cypB) and the target gene (TNF-α) is measured, and the measured value for TNF-α is normalized to cypB to obtain relative luminescence. A unit (relative luminescence unit) was obtained.

  Table 24 summarizes the full-length and truncated knockdown activities of representative polynucleotide delivery-enhancing polypeptide PN73. A “+” in the “Knockdown Activity” column indicates that the peptide / siRNA complex exhibits 80% knockdown activity of the Qneg negative control siRNA (20% decrease in mRNA concentration compared to the Qneg negative control). Indicates what was shown. “+/−” indicates that the peptide / siRNA complex showed about 90% knockdown activity of Qneg negative control siRNA (10% reduction in mRNA concentration compared to Qneg negative control). Finally, “-” indicates that the peptide / siRNA complex did not show significant knockdown activity compared to the Qneg negative control.

  In general, PN643 (full length PN73 not labeled with FITC) and PN690 (full length PN73 labeled with FITC) are “+” in the column of “knockdown activity” (results shown in Table 24). All siRNA tested showed similar siRNA knockdown activity as represented by In addition, PN660 showed siRNA knockdown activity comparable to that of PN643 and PN690 for all siRNA tested, due to the removal of the 9 most N-terminal residues of the PN73 peptide. It shows that the knockdown activity mediated by siRNA against the targeted TNF-α mRNA was not affected. PN654 showed mild knockdown activity for both A19S21 and 21/21 siRNA, but not for LC20 siRNA (knockdown activity is indicated by “±” in the knockdown activity column). ing). However, siRNA complexed to either PN708 or PN735 did not produce observable knockdown activity for any siRNA.

  These results show that the truncated form of the representative polynucleotide delivery-enhancing polypeptide PN73, in particular PN660 and PN654, does not interfere with the ability of siRNA to reduce the mRNA concentration of the target gene, and is effective in human diseases such as RA It has been shown that new methods are provided to improve the delivery of therapeutic siRNA for therapeutic purposes.

Example 19
Characterization of Polynucleotide Delivery-Promoting Polypeptide PN708 In this example, we further explore siRNA cell uptake activity, MFI measurement and knockdown activity of siRNA complexed with PN708 peptide. Available peptides listed in the PN73 deletion series (see Table 23 in Example 17).

  As described above, the cell uptake assay measures the number of cells that have received Cy5-binding siRNA when complexed to a peptide. siRNA cell uptake was measured by flow cytometry. Uptake was expressed as a percentage calculated by dividing the number of cells containing Cy5-binding siRNA by the total number of transfected and untransfected cells in culture. Mean fluorescence intensity (MFI) was measured by flow cytometry, and the amount of Cy5-bound siRNA found in the cells was measured. The MFI value has a positive correlation with the amount of Cy5-binding siRNA in the cell, so a higher MFI value represents a greater number of intracellular Cy5-binding siRNA.

  In this example, the efficiency of siRNA cell uptake activity and MIF measurement were determined using a wide range of peptide concentrations compared to the previous example. In addition, cell viability was also evaluated. In this example, PN643 (full length PN73 with the C-terminal label removed), PN690 (full length PN73 with a FITC label at the C terminus) and PN708 (PN73), which are representative polynucleotide delivery-enhancing polypeptides. 15-mers obtained by deletion of 21 N-terminal residues) were tested at 5 μM, 10 μM, 20 μM and 40 μM. PN643 and PN690 were also tested at 2.5 μM, and PN690 was additionally tested at 1.25 μM. Both PN643 and PN708 were tested at 80 μM.

  As shown in Table 27 below, the non-FITC labeled PN73 (PN643) peptide achieved almost 100% siRNA incorporation at a concentration of 10 μM. However, when the PN73 peptide was labeled with a FITC tag (PN690), its maximum cell uptake activity was reduced to about 70%. PN708 showed a dose-dependent increase in siRNA cell uptake activity. PN708 achieved 95% maximal siRNA cell uptake activity at 80 μM. For full-length PN73 peptide, cell viability decreased with increasing peptide concentration. In contrast, cells incubated with PN708 peptide maintained greater than 90% cell viability in the presence of all concentrations tested. Cy5-MFI measurements further showed that cleaved peptide PN708 effectively doubled the amount of Cy5-siRNA that it delivered to the cells compared to the full-length PN73 (PN690) peptide.

Table 27:

  These results indicate that the cleaved representative polynucleotide delivery-enhancing polypeptide PN768 (residues 34-38 of H2B) allows siRNA to enter the cell without adversely affecting cell viability. It has been shown to have the ability to facilitate highly efficient delivery.

  Further characterization was performed on the cleaved representative polynucleotide delivery-enhancing polypeptide PN708 by measuring its effect on siRNA-mediated reduction of target gene expression. In this section of this example, the FITC label at the C-terminus of the PN708 peptide was removed before assessing its ability to promote reduced target gene expression when complexed with PN708 siRNA. In the absence of FITC label, the cleaved representative polynucleotide delivery-enhancing polypeptide was named PN766 (see Table 23 in Example 17). The ability of the siRNA / peptide complex to modulate the expression of the human tumor necrosis factor-alpha (hTNF-α) gene was evaluated. In this example, random siRNA sequence Qneg served as a negative control, and LC20 and LC17 siRNAs were used to target hTNF-α mRNA in human monocytes. The molar ratio of siRNA to peptide tested was 1: 5; 1:10; 1:25; 1:50; 1:75 and 1: 100. LC20 and LC17 were both used at a concentration of 20 nM.

  The results of the knockdown show that both siRNA / peptide complexes of LC20 / PN766 and LC17 / PN766 at 1: 5; 1:10; and 1:25 show hTNF-α mRNA concentrations, about 70 of the Qneg siRNA negative control. % -80% (ie 20% -30% reduction in mRNA concentration compared to Qneg negative control). The siRNA / peptide ratios of 1:50; 1:75 and 1: 100 did not show a significant effect on hTNF-α mRNA levels compared to the Qneg control. In the presence of PN766 peptide, no cytotoxic effect was observed on human monocytes.

  These data demonstrate that the cleaved representative polynucleotide delivery-enhancing polypeptide PN766, when complexed with siRNA, significantly reduces the mRNA concentration of the target gene. It has been shown to be an ideal siRNA delivery peptide for therapeutic siRNA in the treatment of RA within a subject.

Example 20
Amino acid substitutions and deletions within representative polynucleotide delivery-enhancing polypeptides do not affect peptide-mediated cellular uptake of siRNA This example was created by residue substitutions and / or deletions, Variants of the representative polynucleotide delivery-enhancing polypeptides listed in Table 28 of FIG. 28 affect siRNA cell uptake activity or MFI measurements in comparison to the unmodified representative polynucleotide delivery-enhancing polypeptide PN73. Demonstrate not. Table 28 below lists the residue substitutions made in the representative polynucleotide delivery-enhancing polypeptide PN73. Amino acids highlighted in gray represent unmodified residues, substituted and / or deleted residues. Gray emphasis allows easy identification of unmodified residues in peptide PN73, which can be identified in variants PN644, PN645, PN646, PN647 and PN729 of representative polynucleotide delivery-enhancing polypeptides. Can be easily compared to the substituted and / or deleted residues. Substituted residues within variants of representative polynucleotide delivery-enhancing polypeptides are bolded and underlined. Furthermore, the symbol “^” bolded and underlined represents residues deleted in PN729.

  The purpose of generating representative polynucleotide delivery-enhancing polypeptide variants by residue substitution or deletion was to evaluate the effects of these modifications on siRNA cell uptake activity and the efficiency of siRNA entry into the cell. It was.

Table 28:

  Substitution and / or deletion of specific residues within the representative polynucleotide delivery-enhancing polypeptide PN73 includes changing or increasing the number of aromatic amino acids and / or decreasing the number of negatively charged amino acids. It is. Amino acids with aromatic functional groups (eg, phenylalanine, tyrosine, tryptophan and their derivatives) are typically due to their relatively non-polar (hydrophobic) properties and ability to promote protein cell membrane penetration, Found in the transmembrane domain of proteins. A negatively charged amino acid rejects the negatively charged phosphodiester backbone of the nucleic acid, thus compromising the ability of the protein to bind to the nucleic acid. Thus, the basis for the substitution of an aromatic amino acid within the PN73 peptide includes promoting the cell permeation function of the peptide and / or removal of the negatively charged amino acid causes binding of the protein to the nucleic acid. This is to promote. The ability of the peptide to bind to a nucleic acid can also be facilitated by simply deleting the negatively charged amino acid or replacing the negatively charged amino acid with a positively charged amino acid or a neutral amino acid.

  SiRNA cell uptake assay and MFI measurement were performed as described above. The data is summarized in Table 29 below. Each peptide was tested at concentrations of 0.63 μM, 1.25 μM, 2.5 μM and 5 μM. The results show that, despite the substitution and / or deletion of residues in the representative polynucleotide delivery-enhancing polypeptide PN73, the siRNA cell uptake activity and MFI measurement of the mutant peptide is equivalent to that of the unmodified PN73 It was shown that it was still. These data indicate that residue substitutions and / or deletions did not affect the ability of the peptide to bind to and penetrate the cell membrane. Furthermore, cell viability was not affected by the presence of substituted and / or deleted residues within the representative polynucleotide delivery-enhancing polypeptide PN73.

Table 29:

  These results indicated that, for example, modifications of representative polynucleotide delivery-enhancing polypeptides created by amino acid substitutions or deletions or combinations thereof deliver siRNA with high efficiency to most cells. .

Example 21
Polynucleotide delivery-enhancing polypeptide mediated siRNA cell uptake activity This example demonstrates the effect of siRNA cell uptake activity of the polynucleotide delivery-enhancing polypeptide complexed with siRNA in Table 30. Table 31 summarizes siRNA cell uptake data, mean fluorescence intensity (MFI) measurements and cell viability data for each peptide. Polypeptides that achieve a percent siRNA cell uptake greater than 75% are highlighted in gray in the “Treatment” column. The percent of siRNA cell uptake unique to each of these highlighted siRNA / peptide complexes is also highlighted in gray in the “% siRNA cell uptake” column.

Table 30:

  In the siRNA cell uptake assay in this example, the number of cells that received Cy5-binding LC20 siRNA in the presence of peptide was determined. LC20 is an oligo used for siRNA targeting of human tumor necrosis factor-alpha (hTNF-α) mRNA. Uptake of siRNA by cells was assessed by flow cytometry. Uptake was expressed as a percentage calculated by dividing the number of cells containing Cy5-binding siRNA by the total number of transfected and untransfected cells in culture. Mean fluorescence intensity (MFI) was measured by flow cytometry, and the amount of Cy5-bound siRNA found in the cells was measured. The MFI value has a positive correlation with the amount of Cy5-binding siRNA in the cell, so a higher MFI value represents a greater number of intracellular Cy5-binding siRNA.

The polynucleotide delivery-enhancing polypeptides listed in Table 30 were tested using the following protocol. The day before transfection, approximately 80,000 mouse tail-derived fibroblasts (MTF) per well were seeded in 24-well plates in complete medium. Each delivery peptide except the positive control was tested at concentrations of 0.63 μM, 2.5 μM, 10 μM and 40 μM in the presence of 0.5 μM Cy5-binding siRNA. For siRNA / peptide complexes, Cy5-binding siRNA and peptide were separately diluted in OptiMEM® medium (Invitrogen) to a final concentration of 1/2. Equal amounts of siRNA and peptide were mixed and allowed to complex for 5 minutes at room temperature. The siRNA / peptide complex was then added to cells previously washed with phosphate buffered saline (PBS). Cells were transfected for 3 hours at 37 ° C., 5% CO ₂ . Cells were washed with PBS, treated with trypsin and then analyzed by flow cytometry. SiRNA cell uptake was measured by the intensity of intracellular Cy5 fluorescence. Cell viability was measured by propidium iodide uptake or staining with Annexin V-PE (BD Bioscience). To distinguish cell uptake from insertion of labeled siRNA (or fluorescein labeled peptide) into the membrane, trypan blue was used to quench the fluorescence on the cell membrane surface. Trypan blue (Sigma) was added to the cells to a final concentration of 0.04%, and then flowed through the flow cytometer again to evaluate whether there was any change in the fluorescence intensity indicating fluorescence localized in the cell membrane. did.

Table 31:

  As shown in the column entitled “% siRNA cell uptake” in Table 31, the “untreated” negative control showed no cellular uptake of siRNA, whereas the positive control peptide showed 95% siRNA cell uptake. Percent activity was achieved. Cy5-binding LC20 siRNA complexed with polynucleotide delivery-enhancing polypeptides PN680; PN681; PN709; PN760; PN759 or PN682 achieved a percent siRNA cell uptake activity greater than 75%. Polynucleotide delivery facilitating polypeptides PN694 and PN714 showed moderate siRNA cell uptake activity of 54% and 43%, respectively. In contrast, the polynucleotide delivery-enhancing polypeptides PN665 and PN734 did not show significant siRNA cell uptake activity (less than 5%).

  The polynucleotide delivery-enhancing polypeptide was further characterized by analyzing mean fluorescence intensity (MFI) for its ability to transfect siRNA into cells. In the cell uptake assay, the percentage of cells containing Cy5-binding siRNA was measured, whereas in the MFI measurement, the relative average amount of Cy5-binding siRNA that entered the cells was measured. As shown in the column entitled “siRNA Cy5 MFI” in Table 31, delivery of Cy5 binding siRNA by the positive control peptide PN643 achieved approximately 7 units of MFI. As expected, the “untreated” negative control does not show measurable MFI. The polynucleotide delivery-enhancing polypeptide PN665 was not tested by MFI. PN743, PN694 and PN714 showed significantly lower MFI measurements compared to the positive control. Polynucleotide delivery facilitating polypeptides PN680, PN709 and PN682 showed MFI measurements comparable to the PN643 positive control, while PN681 showed twice the MFI of the positive control. Surprisingly, the polynucleotide delivery-enhancing polypeptides PN760 and PN759 showed MFI measurements about 13 and 6 times higher, respectively, compared to the positive control.

  These data indicate that the polynucleotide delivery-enhancing polypeptides PN680; PN681; PN709; PN760; PN759 and PN682 deliver siRNA efficiently into cells when complexed with siRNA.

Example 22
Gene expression knockdown activity of siRNA transfected into cells using polynucleotide delivery-enhancing polypeptide In this example, siRNA complexed with polynucleotide delivery-enhancing polypeptide reduces the mRNA expression of the target gene of siRNA. Demonstrate effective knockdown. Specifically, the ability of siRNA / peptide complex to regulate the expression of human tumor necrosis factor-α (hTNF-α) gene was evaluated. The implication of targeting the hTNF-α gene is that it is involved in mediating the onset or progression of rheumatoid arthritis (RA) when the hTNF-α gene is overexpressed in humans and other mammalian subjects. It is in thought.

  Human monocytes were used as a model system for measuring the effect of siRNA / peptide complexes on hTNF-α gene expression. Qneg represents a random siRNA sequence and served as a negative control. The observed Qneg knockdown activity is normalized to 100% (100% gene expression level), but the following A19S21 MD8, 21/21 MD8 and LC20 siRNA knockdown activities are relative percentages of the negative control. Expressed as: A19S21 MD8, 21/21 MD8 and LC20 are siRNAs targeting hTNF-α mRNA.

  The polynucleotide delivery-enhancing polypeptide PN602 corresponds to the positive control acetylated form used in the previous examples, and in this example also the effective delivery of siRNA to human monocytes and the siRNA-mediated hTNF -Used as a positive control in both acceptable knockdown activity of the mRNA concentration of α. Polynucleotide delivery-enhancing polypeptides PN680 and PN681 were complexed with the siRNAs listed above to determine the effect of these complexes on the ability of each siRNA to reduce the level of hTNF-α gene expression in human monocytes. . The knockdown activity of all three polynucleotide delivery-enhancing polypeptides is summarized in Table 32 below. A “+” in the “Knockdown Activity” column indicates that the peptide / siRNA complex exhibits 80% knockdown activity of the Qneg negative control siRNA (20% decrease in mRNA concentration compared to the Qneg negative control). Indicates what was shown. “+/−” indicates that the peptide / siRNA complex showed about 90% knockdown activity of Qneg negative control siRNA (10% reduction in mRNA concentration compared to Qneg negative control). Finally, “-” indicates that the peptide / siRNA complex did not show significant knockdown activity compared to the Qneg negative control.

  In this example, transfection in human monocytes was performed according to the protocol described above.

  For mRNA measurement, branch DNA technology from Genospectra (California) was used according to the manufacturer's instructions. In order to quantify the intracellular mRNA concentration, the mRNA of both the housekeeping gene (cypB) and the target gene (TNF-α) is measured, and the measured value for TNF-α is normalized to cypB to obtain relative luminescence. A unit (relative luminescence unit) was obtained.

Table 32:

  The results shown in Table 32 show that all three siRNAs complexed to the positive control PN602 polynucleotide delivery-enhancing polypeptide at ratios of 1: 5 and 1:10 were complexed with the same polypeptide. It shows that hTNF-α gene expression level was gently reduced in comparison with the control. However, the same siRNA complexed with the polynucleotide delivery-enhancing polypeptide PN681 at 1: 5 and 1:10 showed no or equal knockdown activity compared to the Qneg negative control siRNA / PN681 complex. . In contrast, the polynucleotide delivery-enhancing polypeptide PN680 complexed with any of the hTNF-α specific siRNAs at a 1: 5 ratio resulted in significant hTNF-α mRNA knockdown relative to the Qneg / PN680 control complex. Showed activity. In addition, the LC20 / PN680 complex in a 1:10 ratio also showed significant knockdown activity in comparison to the Qneg / PN680 control complex.

  These data indicate that the polynucleotide delivery-enhancing polypeptide PN680 delivers siRNA into cells allowing siRNA-mediated effective gene silencing.

  While the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes or modifications may be practiced within the scope of the appended claims, which are presented by way of illustration and not limitation. It will be clear that it can be done. In this context, various publications and other cited references are cited within the previous disclosure for the sake of saving description. Each of these references is incorporated herein by reference in its entirety for all purposes. However, the various publications discussed herein are incorporated solely for their disclosure prior to the filing date of the present application, and based on the previous invention, such disclosure is prior to the present application. Note that the rights are reserved by the inventor.

FIG. 1 illustrates the effect on peptide-mediated uptake and cell viability by siRNA complexed or bound to the polynucleotide delivery-enhancing polypeptide of the present invention (SEQ ID NO: 35). Cell uptake and cell viability are expressed as percentages. FIG. 2 illustrates peptide-mediated uptake by siRNA complexed or bound to the polynucleotide delivery-enhancing polypeptide of the present invention (SEQ ID NO: 35). Cell uptake is expressed as mean fluorescence intensity (MFI). FIG. 3 shows peptide-mediated uptake of siRNA in human monocytes by a plurality of different polynucleotide delivery-enhancing polypeptides. FIG. 4 illustrates the effect on human monocyte viability after exposure to siRNA complexed with several different polynucleotide delivery-enhancing polypeptides. FIG. 5 shows that mice injected with siRNA / peptide show slower RA progression compared to the RA progression exhibited by remicade treated subjects. RA progression was measured by paw scoring index. FIG. 6 provides the results of tests of uptake efficiency and survival in mouse tail fibroblasts for the polynucleotide delivery-enhancing polypeptide of the present invention, a rationally designed derivative from PN73. FIG. 7 illustrates that peptide-mediated uptake of siRNA complexed with a polynucleotide delivery-enhancing polypeptide of the invention does not cause an interferon response compared to lipofectamine-mediated siRNA delivery. (A): siRNA complexed with lipofectamine (B): siRNA complexed with PN73 (1: 5) FIG. 8 shows that siNA conjugated to a polynucleotide delivery-enhancing polypeptide exhibits stronger knockdown activity in vitro than siRNA complexed with a polynucleotide delivery-enhancing polypeptide. FIG. 9 shows a comparison of cellular uptake between siNA bound to cholesterol and unbound siRNA using a polynucleotide delivery-enhancing polypeptide. FIG. 10 shows that inhibition of cellular uptake of siRNA bound to cholesterol by serum can be rescued by polynucleotide delivery-enhancing polypeptides.

Claims (37)

  A composition comprising a polynucleotide delivery-enhancing polypeptide and a double-stranded ribonucleic acid (dsRNA), wherein the polynucleotide delivery-enhancing polypeptide is amphiphilic and has nucleic acid binding properties.   2. The composition of claim 1, wherein the polynucleotide delivery-enhancing polypeptide comprises about 5 to about 40 amino acids and has a MW 20,000 to 50,000 poly (Lys, Tryp) 4: 1, 20, 000 to 50,000 poly (Orn, Trp) 4: 1, melittin, histone H1, histone H3 and histone H4, SEQ ID NOs: 27 to 31, 35 to 42, 45, 47, 50 to 59, 62, 63, 67 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178, and 180 to 186. .   2. The composition of claim 1, wherein the composition causes dsRNA uptake into animal cells.   4. The composition according to claim 3, wherein the animal cell is a mammalian cell.   The composition of claim 1, wherein the composition is administered to an animal.   6. The composition according to claim 5, wherein the animal is a mammal.   2. The composition according to claim 1, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is acetylated.   The composition according to claim 1, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is PEGylated.   2. The composition of claim 1, wherein the dsRNA comprises a small interfering ribonucleic acid comprising a sequence of about 10 to about 40 base pairs complementary to a portion of a tumor necrosis factor-alpha (TNF-α) gene. A composition characterized in that it is a small interfering ribonucleic acid (siRNA).   2. The composition of claim 1, wherein the dsRNA is a siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID NOs: 109 to 163 and 187. .   2. The composition of claim 1, wherein the polynucleotide delivery-enhancing polypeptide is mixed, complexed or bound with the dsRNA.   2. The composition of claim 1, wherein the polynucleotide delivery-enhancing polypeptide binds to the dsRNA.   The composition according to claim 1, further comprising a cationic lipid.   14. The composition of claim 13, wherein the cationic lipid is N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride, 1,2-bis ( Oleoyloxy) -3-3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2 (Sperminecarboxamido) ethyl] -N, N-dimethyl-1-propananium trifluoroacetic acid, 1,3-dioleoyloxy-2- (6-carboxyspermyl) -propylamide (1,3-dioleoyloxy- 2- (6-carbospermyl) -propylamid), 5 Carboxyspermylglycine dioctadecylamide, tetramethyltetrapalmitoylspermine, tetramethyltetraoleylspermine, tetramethyltetralaurylspermine, tetramethyltetramyristylspermine and tetramethyldioleylspermine, DOTMA (N- [1- (2,3- Dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride), DOTAP (1,2-bis (oleoyloxy) -3,3- (trimethylammonium) propane), DMRIE (1,2-di-) Myristyloxypropyl-3-dimethylhydroxyethylammonium bromide), DDAB (dimethyldioctadecylammonium bromide), polyvalent cationic lipid, lipospermine, DOSPA (2,3-diole) Ruoxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid), DOSPER (1,3-dioleoyloxy-2- (6 carboxyspermyl) propylamide ) (L, 3-dioleoyloxy-2- (6 carboxy spermyl) -propyl-amid), di- and tetra-alkyl-tetra-methyl spermine, TMPPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethyltetralaurylspermine), TTMMS (tetramethyltetramyristylspermine), TMDOS (tetramethyldioleylspermine) DOGS (dioctadecylamide glycylspermine) TRANSFECTAM®, cationic lipids combined with non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol, DOSPA and DOPE 3 A composition characterized in that it is selected from the group consisting of a cationic lipid composition consisting of a 1: 1 (w / w) mixture and a 1: 1 (w / w) mixture of DOTMA and DOPE.   A method for causing uptake of double-stranded ribonucleic acid (dsRNA) into an animal cell, the method comprising incubating the animal cell with a mixture comprising a polynucleotide delivery-enhancing polypeptide and the dsRNA, A method wherein the polynucleotide delivery-enhancing polypeptide is amphiphilic and has nucleic acid binding properties.   A method of altering expression of a target gene in an animal cell, wherein the method is a polynucleotide delivery-enhancing polypeptide, the polynucleotide delivery-enhancing polypeptide being amphiphilic and having nucleic acid binding properties Incubating the animal cell with a mixture comprising a facilitating polypeptide and a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA is complementary to a region of the target gene A method comprising the step of:   The method according to claim 15 or 16, wherein the animal cell is a mammalian cell.   A method for altering the phenotype of a subject animal, wherein the method is a polynucleotide delivery-enhancing polypeptide, the polynucleotide delivery-enhancing polypeptide being amphiphilic and having a nucleic acid binding property A mixture of a double-stranded ribonucleic acid (dsRNA) and a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA is complementary to a target gene region in the subject, A method characterized by comprising.   The method of claim 18, wherein the animal is a mammal.   19. The method of any one of claims 15, 16 or 18, wherein the polynucleotide delivery-enhancing polypeptide comprises about 5 to about 40 amino acids and has a molecular weight of 20,000 to 50,000. , Tryp) 4: 1, 20,000 to 50,000 poly (Orn, Trp) 4: 1, melittin, histone H1, histone H3 and histone H4, SEQ ID NOs: 27 to 31, 35 to 42, 45, 47, 50 to 59, 62, 63, 67, 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178, and 180 to 186. A method characterized by comprising.   The method according to any one of claims 15, 16 or 18, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is acetylated.   The method according to any one of claims 15, 16 and 18, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is PEGylated.   19. The method of any one of claims 15, 16 or 18, wherein the dsRNA is from a sequence of about 10 to about 40 base pairs complementary to a portion of a tumor necrosis factor-alpha (TNF-α) gene. A method comprising small interfering ribonucleic acid (siRNA).   19. The method of any one of claims 15, 16 or 18, wherein the dsRNA is an siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID NOs: 109 to 163 and 187. A method characterized by being.   19. The method according to any one of claims 15, 16 or 18, wherein the polynucleotide delivery-enhancing polypeptide is mixed, complexed or conjugated with the dsRNA. Method.   The method of any one of claims 15, 16 or 18, wherein the polynucleotide delivery-enhancing polypeptide binds to the dsRNA.   19. A method according to any one of claims 15, 16 or 18, further comprising a cationic lipid.   28. The method of claim 27, wherein the cationic lipid is N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride, 1,2-bis (ole). Oiloxy) -3-3- (trimethylammonium) propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2 ( Sperminecarboxamido) ethyl] -N, N-dimethyl-1-propananium trifluoroacetic acid, 1,3-dioleoyloxy-2- (6-carboxyspermyl) propylamide (1,3-dioleoyloxy-2- (6-carbospermyl) -propylamid), 5- Boxyspermylglycine dioctadecylamide, tetramethyltetrapalmitoylspermine, tetramethyltetraoleylspermine, tetramethyltetralaurylspermine, tetramethyltetramyristylspermine and tetramethyldioleylspermine, DOTMA (N- [1- (2,3- Dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride), DOTAP (1,2-bis (oleoyloxy) -3,3- (trimethylammonium) propane), DMRIE (1,2-di-) Myristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide), DDAB (dimethyldioctadecylammonium bromide), polyvalent cationic lipid, lipospermine, DOSPA (2,3-dioleo) Oxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid), DOSPER (1,3-dioleoyloxy-2- (6 carboxyspermyl) propylamide ) (L, 3-dioleoyloxy-2- (6carboxy spermyl) -propyl-amid), di- and tetra-alkyl-tetra-methyl spermine, TMPPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (Tetramethyltetralaurylspermine), TTMMS (Tetramethyltetramyristylspermine), TMDOS (Tetramethyldioleylspermine) DOGS (Dioctadecylamidoglycylspermine (G) TRANSFECTAM®, cationic lipids combined with non-cationic lipids, DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol, DOSPA and DOPE 3: 1 A method characterized in that it is selected from the group consisting of a cationic lipid composition comprising a (w / w) mixture and a 1: 1 (w / w) mixture of DOTMA and DOPE.   A polynucleotide delivery-enhancing polypeptide, wherein the polynucleotide delivery-enhancing polypeptide is amphipathic and has nucleic acid binding properties, and tumor necrosis factor-alpha (TNF-α) of the subject animal ) Double-stranded ribonucleic acid (dsRNA) for the manufacture of a medicament for the treatment of associated inflammatory disorders, wherein the drug reduces TNF-alpha RNA levels, thereby one or more symptoms of TNF-alpha-associated inflammatory disorders Use of a mixture comprising double-stranded ribonucleic acid (dsRNA) capable of preventing or reducing the onset or severity of.   30. The use of the mixture of claim 29, wherein the polynucleotide delivery-enhancing polypeptide comprises about 5 to about 40 amino acids and has a molecular weight of 20,000 to 50,000 poly (Lys, Tryp) 4: 1, 20,000 to 50,000 poly (Orn, Trp) 4: 1, melittin, histone H1, histone H3 and histone H4, SEQ ID NOs: 27 to 31, 35 to 42, 45, 47, 50 to 59, 62, 63 67, 68, 73, 74, 76, 78 to 87, 89 to 92, 94 to 108, 164 to 178, and 180 to 186. .   30. The use of the mixture of claim 29, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is acetylated.   30. The use of the mixture of claim 29, wherein the N-terminus of the polynucleotide delivery-enhancing polypeptide is PEGylated.   30. The use of the mixture of claim 29, wherein the dsRNA consists of a sequence of about 10 to about 40 base pairs complementary to a portion of a tumor necrosis factor-alpha (TNF-α) gene. (Small interfering ribonucleic acid) (siRNA).   30. The use of the mixture of claim 29, wherein the dsRNA is a siRNA consisting of a sequence of about 10 to about 40 base pairs selected from the group consisting of SEQ ID NOs: 109 to 163 and 187. .   30. The use of the mixture of claim 29, wherein the polynucleotide delivery-enhancing polypeptide is mixed, complexed or conjugated with the dsRNA.   30. The use of the mixture of claim 29, wherein the polynucleotide delivery-enhancing polypeptide binds to the dsRNA.   30. Use of the mixture according to claim 29, wherein the subject animal is a mammal.

Images