Functional fragments of HIV-1 VPR protein and methods of using the same

Abstract

Conjugated compositions comprising a fragment of HIV-1 Vpr or a non-HIV-1 Vpr protein conjugated to a therapeutic compounds and methods of using the same to deliver therapeutic compounds to a cell's nucleus or for the preparation of drug delivery particles are disclosed. Functional fragments of HIV-1 Vpr and functional non-HIV-1 Vpr proteins, and pharmaceutical compositions comprising the same are disclosed. Methods of inhibiting cell proliferation and methods of treating an individual who has a hyperproliferative disease are disclosed. Methods of identifying compounds that inhibit Vpr protein binding to the p6 domain of p55 or to p6 protein and kits for performing such methods are disclosed.

1 . A conjugated composition comprising: 
 a fragment of HIV-1 Vpr comprising amino acid sequence 17-36 and/or 59-84 or a non-HIV-1 Vpr protein comprising amino acids amino acids 17-36 and 59-84 conjugated to a therapeutic compound.    
     
     
         2 . The conjugated composition of  claim 1  wherein said fragment of HIV-1 Vpr or said non-HIV-1 Vpr protein further comprises a polycationic amino acid sequence.  
     
     
         3 . The conjugated composition of  claim 1  wherein said therapeutic compound is a DNA vaccine plasmid conjugated to said fragment of HIV-1 Vpr or said non-HIV-1 Vpr protein by ionic bonds.  
     
     
         4 . The conjugated composition of  claim 1  wherein said fragment of HIV-1 Vpr or said non-BV-1 Vpr protein further comprises a polycationic amino acid sequence and said therapeutic compound is a nucleic acid molecule which is conjugated to said polycationic amino acid sequence by ionic bonds.  
     
     
         5 . The conjugated composition of  claim 1  wherein said compound is an antisense molecule.  
     
     
         6 . The conjugated composition of  claim 1  wherein said compound is an antisense oligonucleotide  
     
     
         7 . A method of delivering a compound to the nucleus of a cell comprising the step of: 
 contacting said cell with a conjugated compound that is either said compound conjugated to a fragment of HIV-1 Vpr protein comprising amino acids 17-36 and/or 59-84 or said compound conjugated to a non-HIV-1 Vpr protein comprising amino acids 17-36 and/or 59-84 of HIV-1 Vpr-protein; wherein said conjugated compound is taken up by said cell and localized to the nucleus of said cell.    
     
     
         8 . The method of  claim 7  wherein said compound is a DNA molecule.  
     
     
         9 . The method of  claim 7  wherein said compound is a plasmid DNA molecule.  
     
     
         10 . The method of  claim 7  wherein said compound is an antisense molecule.  
     
     
         11 . The method of  claim 7  wherein said compound is an antisense oligonucleotide  
     
     
         12 . A fragment of HIV-1 Vpr comprising amino acid sequence 17-36 and/or 59-84 or a non-HIV-1 Vpr protein comprising amino acids 17-36 and/or 59-84 of HIV-1 Vpr protein.  
     
     
         13 . A method of inhibiting cell proliferation comprising the step of: 
 arresting said cell's advance in the cell cycle by contacting said cell with 
 a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or  
 a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein; or  
 a nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or  
 a nucleic acid molecule that encodes a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein;  
 wherein said fragment of HIV-1 Vpr or non-HIV-1 Vpr protein is taken up by said cell or  
 said nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein or said nucleic acid molecule that encodes said non-HIV-1 Vpr protein is taken up by said cell and expressed to produce said fragment of HIV-1 Vpr or non-HIV-1 Vpr protein in said cell, and said fragment of HIV-1 Vpr or non-HIV-1 Vpr protein inhibits said cell from advancing in said cell cycle.  
   
     
     
         14 . A method of treating an individual who has a hyperproliferative comprising the step of administering to said individual in an amount effective to inhibit cell proliferation a composition comprising: 
 a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or    a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein; or    a nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or    a nucleic acid molecule that encodes a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein;    wherein said fragment of HIV-1 Vpr or non-HIV-1 Vpr protein molecule is taken up by proliferating cells of said individual or    said nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein or said nucleic acid molecule that encodes said non-HIV-1 Vpr protein is taken up by a proliferating cell of said individual and expressed to produce said fragment of HIV-1 Vpr or non-HIV-1 Vpr protein molecule in said cell, and said fragment of HIV-1 Vpr or non-HIV-1 Vpr protein molecule inhibit said cell from advancing in said cell cycle.    
     
     
         15 . A fragment of HIV-Vpr comprising amino acid sequence or a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV-1 Vpr protein.  
     
     
         16 . A pharmaceutical composition comprising: 
 a fragment of HIV-Vpr or a non-HIV-1 Vpr protein according to  claim 15;  and    a pharmaceutically acceptable carrier.    
     
     
         17 . A nucleic acid molecule that encodes a fragment of HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89; or a nucleic acid molecule that encodes a non-HIV-1 Vpr protein comprising amino acids 19-35 and/or 74-89 of HIV Vpr protein.  
     
     
         18 . A nucleic acid molecule according to  claim 17  wherein said nucleic acid molecule is a plasmid.  
     
     
         19 . A nucleic acid molecule according to  claim 17  wherein said nucleic acid molecule is a viral genome.  
     
     
         20 . A pharmaceutical composition comprising: 
 a nucleic acid molecule according to  claim 17;  and    a pharmaceutically acceptable carrier.    
     
     
         21 . A method of identifying compounds that inhibit Vpr protein binding to the p6 domain of p55 or to p6 protein which comprises the steps of: 
 a) contacting a fragment of HIV-1 Vpr comprising amino acid sequence 17-36 or a non-HIV-1 Vpr protein comprising amino acids 17-36 of HIV-1 Vpr protein with a protein comprising an HIV-1 Gag protein p6 domain in the presence of a test compound,    b) determining the level of binding between said fragment of HIV-1 Vpr or said non-HIV-1 Vpr protein and said protein comprising an HIV-1 Gag p6 domain and    c) comparing that level of binding to the level of binding between said fragment of HIV-1 Vpr or said non-HIV-1 Vpr protein and said protein comprising an HIV-1 Gag p6 domain contacted in the absence of a test compound.    
     
     
         22 . The method of  claim 21  wherein said protein comprising an HIV-1 Gag p6 domain is p55.  
     
     
         23 . The method of  claim 21  wherein said protein comprising an HIV-1 Gag p6 domain is p6.  
     
     
         24 . A kit for performing the method of identifying compounds which inhibit Vpr protein binding to p55's p6 domain or to p6 protein of  claim 21 , said kit comprising: 
 a) a first container comprising a fragment of HIV-1 Vpr comprising amino acid sequence 17-36 or a non-HIV-1 Vpr protein comprising amino acids 17-36 of HIV-1 Vpr protein; and    b) a second container comprising a protein comprising an HIV-1 Gag protein p6 domain.    
     
     
         25 . A fusion protein comprising comprises a Vpr amino acid sequence 17-36 and non-Vpr amino acid sequences.  
     
     
         26 . The fusion protein of  claim 25  wherein said non-Vpr amino acid sequences are biologically active protein sequences.  
     
     
         27 . Drug delivery particles comprising fusion proteins of  claim 25.