Compostions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells

Abstract

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

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