Lentiviral vectors and uses thereof

Abstract

The invention relates to compositions containing polynucleotide vectors capable of expressing a nucleic acid encoding a fusion polypeptide on the surface of a viral particle and/or a eukaryotic cell.

What is claimed is: 
     
         1 . A method of displaying a candidate polypeptide on the surface of a mammalian cell, comprising the steps of:
 a) contacting a packaging cell with a viral display vector comprising a nucleic acid sequence encoding a fusion protein under conditions such that said packaging cell generates one or more viral particles comprising said nucleic acid sequence, wherein said fusion protein comprises:
 i) a leader peptide; 
 ii) a candidate polypeptide; 
 iii) at least a portion of an immunoglobulin molecule; 
 iv) a transmembrane moiety; and 
   b) contacting said viral particles with a mammalian cell, under conditions sufficient to express said fusion protein in said mammalian cell, wherein said candidate polypeptide is displayed on the surface of the mammalian cell.   
     
     
         2 . The method of  claim 1 , wherein said immunoglobulin molecule is further displayed on the surface of the mammalian cell. 
     
     
         3 . The method of  claim 1 , wherein said immunoglobulin molecule comprises the hinge region, the CH2 region or the CH3 region of an IgG molecule or a combination thereof. 
     
     
         4 . The method of  claim 1 , wherein said viral display vector is a lentiviral vector. 
     
     
         5 . The method of  claim 1 , wherein said candidate polypeptide is an antibody or fragment thereof. 
     
     
         6 . The method of  claim 5 , wherein said antibody is a single chain antibody, a domain antibody, a diabody, or Fab fragment. 
     
     
         7 . The method of  claim 1 , wherein said candidate polypeptide is derived from a library. 
     
     
         8 . The method of  claim 4 , wherein said lentiviral vector is obtained from a lentivirus selected from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV), bovine immune deficiency virus (BIV), bovine leukemia virus (BLV), and feline immunodeficiency virus (FIV). 
     
     
         9 . The method of  claim 1 , wherein said packaging cell further comprises;
 (i) an envelope vector comprising an env gene operably linked to a promoter and a polyadenylation sequence wherein said env gene encodes a functional envelope protein and;   (ii) a helper vector comprising one or more viral structural genes.   
     
     
         10 . The method of  claim 1 , wherein said candidate polypeptide is expressed on the surface of said viral particles prior to contacting said viral particles with said mammalian cell. 
     
     
         11 . The method of  claim 1 , wherein said transmembrane moiety is a viral transmembrane moiety or a mammalian transmembrane moiety. 
     
     
         12 . The method of  claim 1 , wherein said mammalian cell is a Ramos Burkitt's lymphoma cell. 
     
     
         13 . A method of displaying a candidate polypeptide on the surface of a viral particle, comprising the step of contacting a packaging cell with a viral display vector comprising a nucleic acid sequence encoding a fusion protein under conditions such that said packaging cell generates one or more viral particles comprising the fusion protein, such that said candidate polypeptide is displayed on the surface of the viral particle, wherein said fusion protein comprises:
 i) a leader peptide;   ii) a candidate polypeptide;   iii) at least a portion of an immunoglobulin molecule;   iv) a transmembrane moiety; and   v) an envelope incorporation signal.   
     
     
         14 . The method of  claim 13 , wherein said candidate polypeptide is an antibody or fragment thereof. 
     
     
         15 . The method of  claim 14 , wherein said antibody is a single chain antibody, a domain antibody, a diabody, or Fab fragment. 
     
     
         16 . The method of  claim 13 , wherein said candidate polypeptide is derived from a library. 
     
     
         17 . The method of  claim 13 , wherein said viral display vector is a lentiviral vector. 
     
     
         18 . The method of  claim 17 , wherein said lentiviral vector is obtained from a lentivirus selected from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV), bovine immune deficiency virus (BIV), bovine leukemia virus (BLV), and feline immunodeficiency virus (FIV). 
     
     
         19 . The method of  claim 13 , wherein said immunoglobulin molecule comprises the hinge region, the CH2 region or the CH3 region of an IgG molecule or a combination thereof. 
     
     
         20 . The method of  claim 13 , wherein said transmembrane moiety is a viral transmembrane moiety or a mammalian transmembrane moiety. 
     
     
         21 . The method of  claim 13 , wherein said packaging cell further comprises;
 (i) an envelope vector comprising an env gene operably linked to a promoter and a polyadenylation sequence wherein said env gene encodes a functional envelope protein and;   (ii) a helper vector comprising one or more viral structural genes.   
     
     
         22 . A method of identifying a candidate polypeptide that binds to a target antigen, comprising the steps of:
 a) contacting a packaging cell with a nucleic acid vector comprising a nucleic acid sequence encoding a fusion protein under conditions such said packaging cell generates one or more viral particles comprising said nucleic acid sequence, wherein said fusion protein comprises:
 i) a leader peptide; 
 ii) a candidate polypeptide; 
 iii) at least a portion of an immunoglobulin molecule; and 
 iv) a transmembrane moiety, and 
   b) contacting said viral particles with one or more mammalian cells, under conditions such that said one or more mammalian cells each generate a single species of said candidate polypeptide displayed on the surface of said mammalian cells;   c) sorting said mammalian cells displaying said single species of said candidate polypeptide on the surface of said mammalian cell;   d) contacting said sorted mammalian cells with a target antigen; and   e) identifying the mammalian cells that bind to said target antigen, thereby identifying a candidate polypeptide that binds to said target antigen.   
     
     
         23 . The method of  claim 22 , further comprising repeating steps d) and e) one or more times. 
     
     
         24 . The method of  claim 22 , wherein said packaging cell further comprises;
 (i) an envelope vector comprising an env gene operably linked to a promoter and a polyadenylation sequence wherein said env gene encodes a functional envelope protein and;   (ii) a helper vector comprising one or more viral structural genes.   
     
     
         25 . The method of  claim 22 , wherein said candidate polypeptide is an antibody or fragment thereof. 
     
     
         26 . The method of  claim 22 , wherein said candidate polypeptide is derived from a library. 
     
     
         27 . The method of  claim 28 , wherein said viral display vector is a lentiviral vector. 
     
     
         28 . The method of  claim 27 , wherein said lentiviral vector is obtained from a lentivirus selected from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV), bovine immune deficiency virus (BIV), bovine leukemia virus (BLV), and feline immunodeficiency virus (FIV). 
     
     
         29 . The method of  claim 22 , wherein said nucleic acid vector further comprises an nucleic acid encoding a selectable marker. 
     
     
         30 . The method of  claim 29 , wherein said selectable marker is selected from the group consisting of a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a blue fluorescent protein, a ferredoxin IV protein, a luciferase protein and a aequorin protein. 
     
     
         31 . The method of  claim 22 , wherein said transmembrane moiety is a viral transmembrane moiety or mammalian transmembrane moiety. 
     
     
         32 . A method of affinity maturing a candidate polypeptide, comprising the steps of:
 a) contacting a packaging cell with a nucleic acid vector comprising a nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises:
 i) a leader peptide; 
 ii) a candidate polypeptide; 
 iii) at least a portion of an immunoglobulin molecule; and 
 iv) a transmembrane moiety under conditions such that the packaging cell generates one or more viral particles comprising said nucleic acid sequence; 
   b) contacting said one or more viral particles with one or more second mammalian cells, under conditions such that said one or more mammalian cells each generate a single species of candidate polypeptide that is displayed on the surface of said one or more mammalian cells;   c) sorting said one or more mammalian cells displaying said single species of candidate polypeptide on the surface of said mammalian cell;   d) contacting said sorted one or more mammalian cells with a target antigen;   e) identifying those mammalian cells that bind to said target antigen;   f) contacting said identified mammalian cells with a decreasing concentration of said target antigen; and   g) repeating steps e) and f) one or more times,   
       thereby affinity maturing a candidate polypeptide that binds to said target antigen. 
     
     
         33 . The method of  claim 32 , wherein said packaging cell further comprises;
 (i) an envelope vector comprising an env gene operably linked to a promoter and a polyadenylation sequence wherein said env gene encodes a functional envelope protein and;   (ii) a helper vector comprising one or more viral structural genes.   
     
     
         34 . The method of  claim 32 , wherein said mammalian cells express activation-induced cytosine deaminase (AID) protein. 
     
     
         35 . The method of  claim 32 , wherein said mammalian cells are Ramos Burkitt's lymphoma cells. 
     
     
         36 . The method of  claim 32 , wherein said candidate polypeptide is derived from a library. 
     
     
         37 . The method of  claim 32 , wherein said viral display vector is a lentiviral vector. 
     
     
         38 . The method of  claim 32 , wherein said candidate polypeptide is an antibody or fragment thereof 
     
     
         39 . The method of  claim 37 , wherein said lentiviral vector is obtained from a lentivirus selected from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV), bovine immune deficiency virus (BIV), bovine leukemia virus (BLV), and feline immunodeficiency virus (FIV). 
     
     
         40 . The method of  claim 37 , wherein said nucleic acid vector further comprises a selectable marker. 
     
     
         41 . The method of  claim 40 , wherein said selectable marker is selected from the group consisting of a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a blue fluorescent protein, a ferredoxin IV protein, a luciferase protein and a aequorin protein. 
     
     
         42 . A method of producing a soluble candidate polypeptide, comprising the step of:
 a) contacting a packaging cell comprising with a viral display vector under conditions such that said packaging cell generates one or more viral particles, wherein said viral vector comprises:
 i) a first a nucleic acid sequence encoding a fusion protein comprising:
 a) a leader peptide; 
 b) a candidate polypeptide; 
 c) at least a portion of an immunoglobulin molecule; and 
 d) a transmembrane moiety flanked by a loxP sites 
 
 ii) a second nucleic acid sequence encoding a cre recombinase polypeptide, operably linked to an inducible promoter, 
   b) contacting said viral particles with a mammalian cell, under conditions sufficient to express said fusion protein and said cre recombinase in said mammalian cell, whereby a soluble candidate polypeptide is produced by said mammalian cell.   
     
     
         43 . The method of  claim 42 , wherein said candidate polypeptide is an antibody. 
     
     
         44 . The method of  claim 43 , wherein said antibody is a single chain antibody, a domain antibody, a diabody, or Fab fragment. 
     
     
         45 . The method of  claim 42 , wherein said candidate polypeptide is derived from a library. 
     
     
         46 . The method of  claim 42 , wherein said viral display vector is a lentiviral vector. 
     
     
         47 . The method of  claim 46 , wherein said lentiviral vector is obtained from a lentivirus selected from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV), bovine immune deficiency virus (BIV), bovine leukemia virus (BLV), and feline immunodeficiency virus (FIV). 
     
     
         48 . A viral display vector comprising a first nucleic acid sequence encoding a fusion protein comprising:
 i) a leader peptide;   ii) a candidate polypeptide;   iii) at least a portion of an immunoglobulin molecule; and   iv) a transmembrane moiety; and   
     
     
         49 . The method of  claim 48 , wherein said immunoglobulin molecule comprises the hinge region, the CH2 region or the CH3 region of an IgG molecule or a combination thereof. 
     
     
         50 . The method of  claim 48 , wherein said candidate polypeptide is an antibody or fragment thereof. 
     
     
         51 . The method of  claim 50 , wherein said antibody is a single chain antibody, a domain antibody, a diabody, or Fab fragment. 
     
     
         52 . The method of  claim 48 , wherein said candidate polypeptide is derived from a library. 
     
     
         53 . The method of  claim 48 , wherein said viral display vector is a lentiviral vector. 
     
     
         54 . The method of  claim 53 , wherein said lentiviral vector is obtained from a lentivirus selected from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV), bovine immune deficiency virus (BIV), bovine leukemia virus (BLV), and feline immunodeficiency virus (FIV). 
     
     
         55 . The viral display vector of  claim 48 , wherein said first nucleic acid further comprises a selectable marker. 
     
     
         56 . The viral display vector of  claim 55 , wherein said selectable marker is a bioluminescent protein moiety. 
     
     
         57 . The viral display vector of  claim 56 , wherein said bioluminescent protein moiety is selected from the group consisting of a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a blue fluorescent protein, a ferredoxin IV protein, a luciferase protein and a aequorin protein. 
     
     
         58 . The viral display vector of  claim 53 , further comprising two or more loxP sites, wherein said loxP sites flank said transmembrane moiety, 
     
     
         59 . The viral display vector of  claim 53 , further comprising a second nucleic acid sequence encoding a cre recombinase protein operably linked to an inducible promoter. 
     
     
         60 . The viral display vector of  claim 53 , wherein said first nucleic acid sequence is operably linked to an inducible promoter. 
     
     
         61 . The viral display vector of  claim 60 , wherein said inducible promoter is selected from the group consisting of a tetracycline inducible promoter, an alcohol inducible promoter, a steroid inducible promoter, a metal inducible promoter, a light inducible promoter, and a temperature inducible promoter. 
     
     
         62 . A vector system, comprising:
 a) the viral display vector of  claim 53 ;   b) an envelope vector comprising an env gene operably linked to a promoter and a polyadenylation sequence wherein said env gene encodes a functional envelope protein and;   (c) a helper vector comprising one or more viral structural genes.   
     
     
         63 . The vector system of  claim 62 , wherein said env gene comprises a vesicular stomatitis virus glycoprotein gene. 
     
     
         64 . The vector system of  claim 62 , wherein said one or more viral structural genes are selected from the group consisting of gag, pol, tat, and rev. 
     
     
         65 . A nucleic acid viral targeting vector comprising:
 a) a first nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises:
 i) a leader peptide; 
 ii) a candidate polypeptide; 
 iii) at least a portion of an immunoglobulin molecule; and 
 iv) a transmembrane moiety flanked by a loxP; 
   b) a second nucleic acid sequence comprising a toxin   c) a third nucleic acid sequence encoding cre recombinase operably linked to an inducible promoter; and   d) a fourth nucleic acid sequence encoding an entry moiety.   
     
     
         66 . The viral targeting vector of  claim 65 , wherein said entry moiety is selected from the group consisting of a viral envelope protein, a eukaryotic protein having fusogenic activity, a binding-defective hemagglutinin protein, and a binding-defective influenza hemagglutinin protein. 
     
     
         67 . A nucleic acid viral targeting vector comprising:
 a) a first nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises:
 i) a leader peptide; 
 ii) a candidate polypeptide; 
 iii) at least a portion of an immunoglobulin molecule; 
 iv) a toxin; and 
 v) a transmembrane moiety flanked by a loxP; 
   b) a second nucleic acid sequence encoding cre recombinase operably linked to an inducible promoter; and   c) a third nucleic acid sequence encoding an entry moiety.   
     
     
         68 . The viral targeting vector of  claim 67 , wherein said entry moiety is selected from the group consisting of a viral envelope protein, a eukaryotic protein having fusogenic activity, a binding-defective hemagglutinin protein, and a binding-defective influenza hemagglutinin protein. 
     
     
         69 . A method for the targeted delivery of a nucleic acid sequence in a subject, wherein said nucleic acid sequence encodes a fusion protein, wherein said fusion protein comprises:
 i) a leader peptide;   ii) a candidate polypeptide;   iii) at least a portion of an immunoglobulin molecule;   iv) a transmembrane moiety; and   v) an entry moiety,   wherein the nucleic acid is expressed on the surface of a viral particle, and wherein the candidate polypeptide specifically binds to a target cell surface antigen, the method comprising introducing said viral particle into the subject, such that said viral particle contacts a cell expressing said target cell surface antigen under conditions such that said candidate polypeptide selectively binds to said cell and said nucleic acid transduces said cell.   
     
     
         70 . The viral targeting vector of  claim 69 , wherein said entry moiety is selected from the group consisting of a viral envelope protein, a eukaryotic protein having fusogenic activity, a binding-defective hemagglutinin protein, and a binding-defective influenza hemagglutinin protein. 
     
     
         71 . The method of  claim 22 , wherein said one or more mammalian cells express a calcium-sensitive bioluminescent protein. 
     
     
         72 . The method of  claim 71 , wherein said calcium-sensitive bioluminescent protein is aequorin.