Nuclear localization signal of lentiviral integrase and method of use thereof

Abstract

The present invention provides novel peptides, nucleic acids, vectors, compounds, compositions and methods for regulating nuclear import The present invention also relates to a lentiviral NLS, and methods of use thereof for inhibiting HIV pathogenesis and disease progression, and for gene delivery methods

1 - 104 . (canceled) 
     
     
         105 . A method of targeting an agent to the nucleus of a cell, comprising the steps of: a) contacting a cell with a lentiviral nuclear localization signal, wherein said lentiviral nuclear localization signal is covalently attached to said agent; and b) culturing said cell under conditions facilitating nuclear import, thereby targeting an agent to the nucleus of a cell. 
     
     
         106 - 107 . (canceled) 
     
     
         108 . The method of  claim 105 , wherein said lentiviral nuclear localization signal is characterized by the formula: R 5 (R 6 ) 2 FrVYR 1 R 3 R 2 R 8 R 3 nplWR 3 GPR 8 kR 4 R 5 WR 3 GEGA(R 4 ) 3 R 6 dR 6 sR 6 R 4 kvR 4 PR(R 3 ) 2 R 8 KIR 4 R 3 d, wherein:
 R 1 =an aromatic amino acid   R 2 =an acidic amino acid   R 3 =a basic amino acid   R 4 =a branched chain amino acid   R 5 =a hydrophobic amino acid   R 6 =a polar amino acid   R 7 =a praline or serine amino acid residue   R 8 =a small amino acid residue   
       lower case letters=the preferred amino acid residue, however substitutions with amino acid residues from disparate groups are possible as well. 
     
     
         109 . The method of  claim 105 , wherein said lentiviral nuclear localization signal comprises an amino acid sequence corresponding to or at least 75% homologous to SEQ ID NO: 139, 147 or 149. 
     
     
         110 . The method of  claim 105 , wherein said nucleic acid encoding for a lentiviral nuclear localization signal has a nucleotide sequence corresponding to or at least 75% homologous to SEQ ID NO: 130, 132, 138 or 148. 
     
     
         111 - 117 . (canceled) 
     
     
         118 . The method of  claim 105 , wherein said agent is further modified to comprise a detectable marker. 
     
     
         119 . The method of  claim 105 , wherein said agent or said lentiviral localization signal is further modified to comprise a targeting moiety. 
     
     
         120 . The method of  claim 119 , wherein said targeting moiety is an antibody or antibody fragment. 
     
     
         121 . The method of  claim 119 , wherein said targeting moiety is a ligand for a receptor expressed on the surface of a cell wherein said targeting is desired. 
     
     
         122 . The method of  claim 119 , wherein said targeting moiety is a receptor for a ligand expressed on the surface of a cell wherein said targeting is desired. 
     
     
         123 . The method of  claim 119 , wherein said targeting moiety is cleaved following uptake of said recombinant protein within a cell. 
     
     
         124 . A method of targeting a protein of interest to the nucleus of a cell, comprising the steps of: contacting a cell with a vector comprising a nucleic acid encoding a lentiviral nuclear localization signal, wherein said nucleic acid encoding said lentiviral nuclear localization signal is fused in frame to a nucleic acid encoding a protein of interest, thereby targeting a protein of interest to the nucleus of a cell. 
     
     
         125 . The method of  claim 124 , wherein said lentiviral nuclear localization signal is characterized by the formula: R 5 (R 6 ) 2 FrVYR 1 R 3 R 2 R 8 R 3 nplWR 3 GPR 8 kR 4 R 5 WR 3 GEGA(R 4 ) 3 R 6 dR 6 sR 6 R 4 kvR 4 PR(R 3 ) 2 R 8 KIR 4 R 3 d, wherein:
 R 1 =an aromatic amino acid   R 2 =an acidic amino acid   R 3 =a basic amino acid   R 4 =a branched chain amino acid   R 5 =a hydrophobic amino acid   R 6 =a polar amino acid   R 7 =a praline or serine amino acid residue   R 8 =a small amino acid residue   
       lower case letters=the preferred amino acid residue, however substitutions with amino acid residues from disparate groups are possible as well. 
     
     
         126 . The method of  claim 124 , wherein said lentiviral nuclear localization signal comprises an amino acid sequence corresponding to or at least 75% homologous to SEQ ID NO: 139, 147 or 149. 
     
     
         127 . The method of  claim 124 , wherein said nucleic acid encoding for a lentiviral nuclear localization signal has a nucleotide sequence corresponding to or at least 75% homologous to SEQ ID NO: 130, 132, 138 or 148. 
     
     
         128 - 134 . (canceled) 
     
     
         135 . The method of  claim 124 , wherein said vector further comprises a nucleic acid encoding for a detectable marker. 
     
     
         136 . The method of  claim 124 , wherein said nucleic acid encoding for a detectable marker is fused in frame to said nucleic acid encoding a protein of interest at a site distal to that of the nucleic acid encoding said lentiviral nuclear localization signal. 
     
     
         137 . The method of  claim 124 , wherein said protein of interest regulates gene expression. 
     
     
         138 . The method of  claim 137 , wherein said protein of interest regulates gene expression by stimulating or enhancing transcription of said gene. 
     
     
         139 . The method of  claim 138 , wherein said protein regulates gene expression by repressing or diminishing transcription of said gene. 
     
     
         140 . The method of  claim 124 , wherein said protein of interest is cytotoxic. 
     
     
         141 . The method of  claim 124 , wherein said protein of interest stimulates or enhances apoptosis. 
     
     
         142 - 186 . (canceled) 
     
     
         187 . The method of  claim 124 , wherein said vector is a viral particle.