Lentivirus-based gene transfer vectors

Abstract

A recombinant lentiviral vector expression system comprises a first vector that comprises a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome. The vector contains at least one defect in at least one gene encoding an EIAV structural protein, but is preferably a gag/pol expression vector. The expression system further comprises a second vector, also comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, and additionally containing a multiple cloning site wherein a heterologous gene may be inserted. The expression system also comprises a third vector which expresses a viral envelope protein. The first and third vectors are packaging signal-defective. When the expression system is transfected into a lentivirus-permissive cell, replication-defective EIAV particles may be produced, which particles are useful in delivering heterologous genes to a broad range of both dividing and non-dividing cells.

That which is claimed:  
     
         1 . A recombinant lentiviral vector expression system comprising: 
 (a) a first vector comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, wherein said vector (i) contains at least one defect in at least one gene encoding an EIAV structural protein, and (ii) contains a defective packaging signal;    (b) a second vector comprising a nucleic acid sequence of at least part of the EIAV genome, wherein said vector (i) contains a competent packaging signal, and (ii) contains a multiple cloning site wherein a heterologous gene may be inserted; and    (c) a third vector comprising a nucleic acid sequence of a virus, wherein said third vector (i) expresses a viral envelope protein, and (ii) contains a defective packaging signal.    
     
     
         2 . A vector system according to  claim 1 , wherein said second vector is deficient for expression of at least one EIAV structural protein.  
     
     
         3 . A vector system according to  claim 1 , wherein said first vector, said second vector, and said third vector are cDNA clones of at least part of the EIAV genome.  
     
     
         4 . A vector expression system according to  claim 1 , wherein said first vector is a gag-pol expression vector, and wherein said vector contains a defect in the env gene.  
     
     
         5 . A vector expression system according to  claim 4 , wherein said defect in the env gene is a deletion mutation.  
     
     
         6 . A vector expression system according to  claim 1 , wherein said first vector and said second vector each contain a defect in the env gene.  
     
     
         7 . A vector expression system according to  claim 1 , wherein said third vector encodes an envelope protein that is not an EIAV envelope protein.  
     
     
         8 . A vector expression system according to  claim 1 , wherein said third vector expresses the vesicular stomatitis virus G glycoprotein.  
     
     
         9 . A vector expression system according to  claim 1 , wherein said second vector contains a heterologous gene.  
     
     
         10 . A vector expression system according to  claim 9 , wherein said heterologous gene encodes an antigenic protein or peptide.  
     
     
         11 . A vector expression system according to  claim 1 , wherein said first vector is selected from the group consisting of the plasmid pEV53 and the plasmid pEV53A; said second vector is selected from the group consisting of pEC-lacZ and pEC-puro; and said third vector is the plasmid pCI-VSV-G.  
     
     
         12 . The plasmid set forth in FIG. 2A as pEV53.  
     
     
         13 . The plasmid set forth in FIG. 2B as pEV53A.  
     
     
         14 . The plasmid set forth-in FIG. 2 as pEC lacZ.  
     
     
         15 . The plasmid set forth in FIG. 3 as pEC-puro.  
     
     
         16 . The plasmid set forth in FIG. 4 as pCI-VSV-G.  
     
     
         17 . A method of producing a replication-defective lentivirus particle, comprising transfecting a cell with: 
 (a) a first vector comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, wherein said vector (i) contains at least one defect in at least one gene encoding an EIAV structural protein, and (ii) contains a defective packaging signal;    (b) a second vector comprising a nucleic acid sequence of at least part of the EIAV genome, wherein said vector (i) contains a competent packaging signal, and (ii) contains a multiple cloning site wherein a heterologous gene may be inserted; and    (c) a third vector-comprising a nucleic acid sequence of a virus, wherein said third vector (i) expresses a viral envelope protein, and (ii) contains a defective packaging signal.    
     
     
         18 . A method according to  claim 17 , wherein said cell is a non-dividing cell.  
     
     
         19 . A method according to  claim 17 , wherein said second vector contains a heterologous gene.  
     
     
         20 . A replication-defective lentivirus particle produced according to the method of  claim 17 .  
     
     
         21 . An infectious EIAV particle containing an EIAV nucleic acid sequence encoding a promoter and a heterologous gene sequence, and wherein said nucleic acid sequence is defective in encoding at least one EIAV structural protein so that said virus particle is replication defective.  
     
     
         22 . A cell containing a replication-defective lentiviral particle, wherein said lentiviral particle is produced according to the method of  claim 17 .  
     
     
         23 . A method of delivering a heterologous gene to a target cell, comprising transfecting said target cell with: 
 (a) a first vector comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, wherein said vector (i) contains at least one defect in at least one gene encoding an EIAV structural protein, and (ii) contains a defective packaging signal;    (b) a second vector comprising a nucleic acid sequence of at least part of the EIAV genome, wherein said vector (i) contains a competent packaging signal, and (ii) contains a multiple cloning site wherein a heterologous gene may be inserted; and    (c) a third vector comprising a nucleic acid sequence of a virus, wherein said third vector (i) expresses a viral envelope protein, and (ii) contains a defective packaging signal.    
     
     
         24 . A method according to  claim 23 , wherein said target cell is a non-dividing cell.  
     
     
         25 . A method of producing a lentiviral stock comprising: 
 (a) transfecting a lentivirus-permissive producer cell with 
 (i) a first vector comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, wherein said vector (1) contains at least one defect in at least one gene encoding an EIAV structural protein, and (2) contains a defective packaging signal;  
 (ii) a second vector comprising a nucleic acid sequence of at least part of the EIAV genome, wherein said vector (1) contains a competent packaging signal, (2) contains a heterologous gene; and  
 (iii) a third vector comprising a nucleic acid sequence of a virus, wherein said third vector (1) expresses a viral envelope protein, and (2) contains a defective packaging signal;  
   (b) growing said producer cell under cell culture conditions sufficient to allow production of replication-defective lentivirus particles in said cell; and    (c) collecting said replication-defective lentivirus particles from said producer cell.    
     
     
         26 . A method according to  claim 25 , wherein said producer cell is grown in a cell culture medium, and wherein said replication-defective lentivirus particles are collected from said medium.  
     
     
         27 . A method of delivering a heterologous gene to a target cell, comprising infecting said target cell with replication-defective lentivirus particles collected according to step (c) of  claim 25 .  
     
     
         28 . A method according to  claim 27 , wherein said target cell is a non-dividing cell.  
     
     
         29 . A method according to  claim 27 , wherein said target cell is a human airway epithelial cell.  
     
     
         30 . A method of delivering a heterologous gene to a target cell, comprising infecting said target cell with a replication-defective lentivirus particle according to  claim 20  or  21 .  
     
     
         31 . A pharmaceutical formulation comprising a replication-defective lentivirus particle according to  claim 20  or  claim 21  in a pharmaceutically acceptable carrier.  
     
     
         32 . A method of making a packaging cell, comprising transfecting a lentivirus-permissive cell with a vector comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, wherein said vector contains a defective packaging signal.  
     
     
         33 . A method according to  claim 32 , wherein said vector is a gal-pol expression vector.  
     
     
         34 . A method according to  claim 32 , wherein said vector is selected from the group consisting of the plasmid pEV53 and the plasmid pEV53A.  
     
     
         35 . A method according to  claim 32 , wherein said lentivirus-permissive cell is a human 293 cell.  
     
     
         36 . A packaging cell comprising a lentivirus-permissive host cell containing an EIAV nucleic acid sequence encoding at least one EIAV structural protein, wherein said nucleic acid sequence is packaging-signal defective, such that the cell itself is capable of producing at least one EIAV structural protein, but not capable of producing replication-competent infectious virus.