High efficiency genetic modification methods

Abstract

A method is provided for producing a population of genetically modified T cells. In the method, an in vitro population of T cells is activated by contacting said population with a CD3 binding agent. Genetic modification is then carried out with the activated T cells by contacting the same with a suitable gene transfer vector.

We claim:  
     
         1 . A method for producing a population of transduced T cells, said method comprising: 
 (a) providing an in vitro population of T cells;    (b) activating the T cells by contacting said population with a CD3 binding agent; and    (c) transducing activated T cells obtained in step (b) by contacting said T cells with a suitable gene transfer vector, wherein transduction is carried out when the cell density of the T cell population is between about 0.1×10 6  and 5×10 6 .    
     
     
         2 . The method of    claim 1   , wherein transduction in step (c) is carried out when the cell density of the T cell population is between about 0.5×10 6  and 2×10 6.    
     
     
         3 . The method of    claim 1   , wherein the gene transfer vector comprises a promoter operably linked to a first nucleotide sequence capable of being expressed to provide a transduced cell with enhanced susceptibility to a selected cytotoxic agent.  
     
     
         4 . The method of    claim 3   , wherein the first nucleotide sequence is a suicide gene.  
     
     
         5 . The method of    claim 4   , wherein the first nucleotide sequence is a Herpes Simplex Virus thymidine kinase (HSV-tk) gene.  
     
     
         6 . The method of    claim 1   , wherein the gene transfer vector further comprises a second nucleotide sequence encoding a selectable marker.  
     
     
         7 . The method of    claim 6   , wherein the selectable marker is capable of providing a transduced T cell with resistance to a selected cytotoxic agent.  
     
     
         8 . The method of    claim 7   , wherein the selectable marker is neomycin phosphotransferase II.  
     
     
         9 . The method of    claim 6   , wherein the selectable marker is a cell surface marker.  
     
     
         10 . The method of    claim 5   , wherein the gene transfer vector further comprises a selectable marker.  
     
     
         11 . The method of    claim 10   , wherein the selectable marker is neomycin phosphotransferase II.  
     
     
         12 . The method of    claim 7   , further comprising a selection step which comprises: contacting the T cells obtained after step (c) with the selected cytotoxic agent, whereby non-transduced T cells can be negatively selected away from the population.  
     
     
         13 . The method of    claim 9   , further comprising a selection step which comprises: contacting the T cells obtained after step (c) with a binding molecule specific for the cell surface marker, whereby transduced T cells can be positively selected away from the population.  
     
     
         14 . The method of    claim 1   , further comprising a selection step which comprises fluorescence-activated cell sorting (FACS) of the T cells obtained after step (c), whereby non-transduced T cells can be separated from transduced T cells.  
     
     
         15 . The method of    claim 13   , wherein the selection step comprises fluorescence-activated cell sorting (FACS) of the T cells obtained after step (c).  
     
     
         16 . The method of    claim 1   , wherein the gene transfer vector is a retroviral vector.  
     
     
         17 . The method of    claim 5   , wherein the gene transfer vector is a retroviral vector.  
     
     
         18 . The method of    claim 1   , wherein the T cell population is contacted with the CD3 binding agent in step (b) for 3 to 4 days.  
     
     
         19 . The method of    claim 1   , wherein the CD3 binding agent is an antibody molecule specific for CD3.  
     
     
         20 . The method of    claim 20   , wherein the antibody molecule is an OKT-3 antibody.  
     
     
         21 . The method of    claim 1   , wherein transduction in step (c) is carried out with a viral vector at a multiplicity of infection (MOI) of about 3 or greater.  
     
     
         22 . A method for producing a population of transduced T cells, said method comprising: 
 (a ) providing an in vitro population of T cells;    (b) activating the T cells by contacting said population with a CD3 binding agent and a mitogen; and    (c) transducing activated T cells obtained in step (b) by contacting said T cells with a suitable gene transfer vector, where in transduction is carried out when the cell density of the T cell population is between about 0.1×10 6  and 5×10 6 .    
     
     
         23 . The method of    claim 22   , wherein the mitogen in step (b) is a cytokine.  
     
     
         24 . The method of    claim 23   , wherein the cytokine is IL-2.  
     
     
         25 . The method of    claim 24   , wherein the IL-2 contacted with the population of T cells in step (b) is added to the population at a concentration of about 50 to 100 μg/mL.  
     
     
         26 . The method of    claim 22   , wherein transduction in step (c) is carried out when the cell density of the T cell population is between about 0.5×10 6  and 2×10 6 .  
     
     
         27 . The method of    claim 22   , wherein the gene transfer vector comprises a first nucleotide sequence capable of being expressed to provide a transduced cell with enhanced susceptibility to a selected cytotoxic agent.  
     
     
         28 . The method of    claim 27    wherein the first nucleotide sequence is a suicide gene.  
     
     
         29 . The method of    claim 28   , wherein the first nucleotide sequence is a Herpes Simplex Virus thymidine kinase (HSV-tk) gene.  
     
     
         30 . The method of    claim 22   , wherein the gene transfer vector is a retroviral vector.  
     
     
         31 . The method of    claim 30   , wherein the retroviral vector is added to the T cell population in step (c) at a multiplicity of infection (MOI) of about 3 or greater.  
     
     
         32 . A method for producing a population of transduced T cells, said method comprising: 
 (a) providing an in vitro population of T cells;    (b) activating the T cells by contacting said population with a CD3 binding agent;    (c) washing the T cell population obtained in step (b) and re-seeding the T cells at a cell density of about 5×10 5 ; and    (d) transducing the T cell population obtained in step (c) by contacting said T cells with a suitable gene transfer vector, wherein transduction is carried out when the cell density of the T cell population is between about 5×10 5  and 2×10 6 .    
     
     
         33 . The method of    claim 32   , wherein the gene transfer vector comprises a first nucleotide sequence capable of being expressed to provide a transduced cell with enhanced susceptibility to a selected cytotoxic agent.  
     
     
         34 . The method of    claim 33    wherein the first nucleotide sequence is a suicide gene.  
     
     
         35 . The method of    claim 34   , wherein the first nucleotide sequence is a Herpes Simplex Virus thymidine kinase (HSV-tk) gene.  
     
     
         36 . The method of    claim 32   , wherein the gene transfer vector is a retroviral vector.  
     
     
         37 . A method for obtaining a transduction efficiency of 100% or greater in a nonselected population of transduced T cells, said method comprising: 
 (a) providing an in vitro population of T cells;    (b) activating the T cells by contacting said population with a CD3 binding agent;    (c) transducing activated T cells obtained in step (b) by contacting said T cells with a retroviral vector at a multiplicity of infection (MOI) of about 3 or greater, wherein transduction is carried out when the cell density of the T cell population is between about 5×10 5  and 2×10 6 .    
     
     
         38 . The method of    claim 37   , wherein the retroviral vector comprises a first nucleotide sequence capable of being expressed to provide a transduced cell with enhanced susceptibility to a selected cytotoxic agent.  
     
     
         39 . The method of    claim 38    wherein the first nucleotide sequence is a suicide gene.  
     
     
         40 . The method of    claim 39   , wherein the first nucleotide sequence is a Herpes Simplex Virus thymidine kinase (HSV-tk) gene.  
     
     
         41 . A kit for producing a population of transduced T cells, said kit comprising a CD3 binding agent contained in one or more containers, a gene transfer vector contained in one or more containers, ancillary reagents and/or hardware, and instructions for use of the kit.  
     
     
         42 . The kit of    claim 41   , wherein the CD3 binding agent is an antibody molecule specific for CD3.  
     
     
         43 . The kit of    claim 42   , wherein the antibody molecule is an OKT-3 antibody.  
     
     
         44 . The kit of    claim 41   , wherein the gene transfer vector comprises a promoter operably linked to a first nucleotide sequence capable of being expressed to provide a transduced cell with enhanced susceptibility to a selected cytotoxic agent.  
     
     
         45 . The kit of    claim 44    wherein the first nucleotide sequence is a suicide gene.  
     
     
         46 . The kit of    claim 45   , wherein the first nucleotide sequence is a Herpes Simplex Virus thymidine kinase (HSV-tk) gene.  
     
     
         47 . The kit of    claim 41   , wherein the gene transfer vector further comprises a second nucleotide sequence encoding a selectable marker.  
     
     
         48 . The kit of    claim 47   , wherein the selectable marker is capable of providing a transduced T cell with resistance to a selected cytotoxic agent.  
     
     
         49 . The kit of    claim 48   , wherein the selectable marker is neomycin phosphotransferase II.  
     
     
         50 . The kit of    claim 48   , wherein the selectable marker is a cell surface marker.