Methods and compositions for genetically modifying primate bone marrow cells

Abstract

A method is provided for genetically modifying primate bone marrow cells, comprising isolating bone marrow cells from a primate and, by means which enhance the local concentration of retroviral particles, contacting the isolated bone marrow cells to cells that produce a recombinant amphotropic retrovirus with a genome based on a retroviral vector that contains the genetic information to be introduced into the bone marrow cells. Recombinant amphotropic retrovirus-producing cells, suitable for use in this method also are provided, as are genetically modified primate bone marrow cells with the capacity for regeneration in vivo.

What is claimed is:  
     
         1 . A method for genetically modifying primate hematopoietic stem cells, said method comprising: 
 combining isolated primate hematopoietic stem cells which can undergo replication with a recombinant retrovirus comprising a genome based on a retroviral vector into which a gene X is inserted, wherein said combining uses means whereby said hematopoietic stem cells and said recombinant retrovirus are brought into close physical contact, so that chance of infection of said hematopoietic stem cells by said recombinant retrovirus is significantly enhanced.    
     
     
         2 . The method according to  claim 1 , wherein said means comprises co-cultivation of said hematopoietic stem cells with cells which produce said recombinant retrovirus.  
     
     
         3 . The method according to  claim 1 , wherein said means are physical means.  
     
     
         4 . The method according to  claim 3 , wherein said physical means comprise centrifugation of medium containing said hematopoietic stem cells and said recombinant retrovirus.  
     
     
         5 . The method according to  claim 1 , wherein said means comprise use of a compound which has binding affinity for both said recombinant retrovirus and said hematopoietic stem cells.  
     
     
         6 . The method according to  claim 5 , wherein said compound which has binding affinity for′both hematopoietic stem cells and said recombinant retrovirus is a component of an extracellular matrix present in a natural hematopoietic environment.  
     
     
         7 . The method according to  claim 1 , wherein said retroviral vector is derived from a viral MuLV vector.  
     
     
         8 . The method according to  claim 7 , wherein said retroviral vector comprises as operably linked components with said gene X, a 5′ LTR and a modified 3′ LTR, wherein said 5˜LTR and said 3′ LTR are derived from a ,Jiral MuLV vector, and a 5′ part of a MuLV gag gene.  
     
     
         9 . The method according to  claim 8 , wherein said MuLV vector is derived from a Mo-MuL¥ vector and wherein said modified 3′ LTR is a hybrid LTR that contains a PyF101 enhancer instead of a Mo-MuLV enhancer.  
     
     
         10 . The method according to  claim 9 , wherein said retroviral vector comprises vector pLgXL(AMo+PyFI01).  
     
     
         11 . The method according to  claim 2 , wherein said cells which produce said recombinant retrovirus are mammalian cells which express MuLV gag, pol and env genes.  
     
     
         12 . The method according to  claim 11 , wherein said env gene is derived from an amphotropic MuLV.  
     
     
         13 . The method according to  claim 11 , wherein said MuLV gag, poi and env genes are contained in at least two different eukaryotic expression vectors.  
     
     
         14 . The method according to  claim 11 , wherein each of said expression vectors is associated with a selectable marker gene.  
     
     
         15 . The method according to  claim 11 , wherein said mammalian cells are GP+envAM 12 cells.  
     
     
         16 . The method according to  claim 10  wherein said retrovirus is an amphotropic retrovirus.  
     
     
         17 . The method according to  claim 16 , wherein cells which produce said recombinant amphotropic retrovirus contain several copies of said retroviral vector.  
     
     
         18 . The method according to  claim 2 , wherein said cocultivation is in medium which contains serum and at least one hematopoietic growth factor.  
     
     
         19 . The method according to  claim 2 , wherein following said co-cultivation, non-adherent bone marrow cells are harvested together with adherent bone marrow cells.  
     
     
         20 . A cell which produces a recombinant retrovirus comprising a genome based on a retroviral vector into which is inserted a gene X for introduction into isolated primate bone marrow cells by the method of cocultivating isolated primate bone marrow cells with said recombinant retrovirus.  
     
     
         21 . The cell of  claim 20 , wherein said retroviral vector is derived from a viral MuLV vector.  
     
     
         22 . The cell of  claim 21 , wherein said retroviral vector comprises as operably linked components with said gene X, a 5′ LTR and a modified 3′ LTR, wherein said 5′ LTR and said 3′ LTR are derived from a viral MuLV vector, and a 5′ part of a MuLV gag gene.  
     
     
         23 . The cell of  claim 22 , wherein said viral MuLV vector is derived from a viral Mo-MuLV vector and said modified 3′ LTR is a hybrid LTR that contains a PyF101 enhancer instead of a Mo-MuLV enhancer.  
     
     
         24 . The cell of  claim 23 , wherein said retroviral vector is pLgXL(&Mo+PyF101), wherein X represents said gene X.  
     
     
         25 . The cell of  claim 18 , wherein said retrovims is an amphotropic retrovirus.  
     
     
         26 . The cell of  claim 25 , wherein said cell is a mammalian cell which expresses MuLV gag, pol and env genes.  
     
     
         27 . The cell of  claim 26 , wherein said env gene is derived from an amphotropic MuLV.  
     
     
         28 . The cell of  claim 26 , wherein said MuLV gag, poi and env genes are contained in at least two different eukaryotic expression vectors.  
     
     
         29 . The cell of  claim 26 , wherein each of said expression vectors are associated with a selectable marker gene.  
     
     
         30 . The cell of  claim 26 , wherein said cells are GP+envAM]2 cells.  
     
     
         31 . The cell of  claim 26 , wherein said recombinant retrovirus contains several copies of said retroviral vector.  
     
     
         32 . A process for producing genetically modified primate hematopoietic cells with long term repopulating capacity, said method comprising: 
 collecting both adherent and nonadherent genetically modified primate bone marrow cells from a culture in which unmodified primate bone marrow cells were combined with a recombinant retrovirus under conditions whereby retroviral particle concentration in the immediate vicinity of said unmodified bone marrow cells was enhanced so as to increase efficiency of transduction of said bone marrow cells.    
     
     
         33 . Genetically modified primate hematopoietic cells with long term repopulating capacity comprising: 
 a plurality of adherent and nonadherent genetically modified primate bone marrow cells.    
     
     
         34 . Genetically modified primate hematopoietic produced by the method of  claim 1 .  
     
     
         35 . A method for genetically modifying primate hematopoietic stem cells, said method comprising combining isolated primate hematopoietic stem cells which can undergo replication in culture with a recombinant retrovirus comprising a genome based on a retroviral vector into which a gene X is inserted, wherein said combining uses means whereby said hematopoietic stem cells and said recombinant retrovims are brought into close physical contact, so that chance of infection of said hematopoietic stem cells by said recombinant retrovirus is significantly enhanced.  
     
     
         36 . The method according to  claim 35 , wherein said replication is stimulated by adding recombinant hematopoietic growth factors to said culture.  
     
     
         37 . The method according to  claim 35 , wherein said culture includes a stromal cell support and replication is stimulated by hematopoietic growth factors produced by said stromal cell support.  
     
     
         38 . The method according to  claim 35 , wherein said combining is cocultivating with a cell line which produces said recombinafit retrovirus and wherein said replication is stimulated by hematopoietic growth factors produced by said cell line.  
     
     
         39 . A cell culture comprising: 
 isolated primate hematopoietic stem cells which can undergo replication and a retrovims producing cell line which produces a recombinant retrovirus comprising a genome based on a retroviral vector into which a gene X is inserted.    
     
     
         40 . The cell culture of  claim 39 , further comprising a stromal cell.  
     
     
         41 . The method according to  claim 5 , wherein said compound which has binding affinity for both hematopoietic stem cells and said recombinant retrovirus is a component present in a natural hematopoietic environment.  
     
     
         42 . The method according to  claim 5 , wherein said compound which has binding affinity for both hematopoietic stem cells and said recombinant retrovirus is a component of cells derived from a natural hematopoietic environment.  
     
     
         43 . A method for genetically modifying primate hematopoietic stem cells, said method comprising: 
 combining isolated primate hematopoietic stem cells which can undergo replication with a recombinant retrovirus comprising a genome based on a retroviral vector into which a gene X is inserted, wherein said combining uses means whereby said hematopoietic stem cells and said recombinant retrovirus are brought into close physical contact, so that chance of infection of said hematopoietic stem cells by said recombinant retrovirus is significantly enhanced.    
     
     
         44 . A cell culture comprising: isolated primate hematopoietic stem cells which can undergo replication and a stromal cell population, wherein said stromal cell population comprises cultured cells, extracellular matrix molecules and cytokines produced by said cultured cells.  
     
     
         45 . The cell culture according to  claim 44 , wherein said stromal cell population is derived from human bone marrow mononuclear cells.