Retroviral Vector

Abstract

A cell for producing retroviral vectors comprising nucleic acid sequences encoding: i) gag-pol; ii) env; iii) the RNA genome of the retroviral vector; and iv) optionally rev, or a functional substitute thereof, wherein at least two nucleic acid sequences are located at the same genetic locus; and wherein the at least two nucleic acid sequences are in reverse and/or alternating orientations.

1 . A cell comprising nucleic acid sequences encoding:
 i) gag-pol;   ii) env;   iii) optionally the RNA genome of the retroviral vector; and   iv) optionally rev,   
       wherein at least two of the nucleic acid sequences are located at the same genetic locus; wherein the at least two of the nucleic acid sequences are in reverse and/or alternating orientations relative to one another; wherein at least one of the nucleic acid sequences encoding gag-pol or env is associated with at least one regulatory element; and wherein the cell is capable of producing a retroviral vector. 
     
     
         2 . A cell comprising nucleic acid sequences encoding:
 i) gag-pol;   ii) env;   iii) optionally the RNA genome of the retroviral vector; and   iv) rev,   
       wherein at least two of the nucleic acid sequences are located at the same genetic locus; wherein the nucleic acid sequences encoding rev and env are in reverse and/or alternating orientations relative to one another; and wherein the cell is capable of producing a retroviral vector. 
     
     
         3 . A transient production cell comprising nucleic acid sequences encoding:
 i) gag-pol;   ii) env;   iii) optionally the RNA genome of the retroviral vector; and   iv) rev,   
       wherein at least two of the nucleic acid sequences are located at the same genetic locus; wherein the nucleic acid sequences encoding gag-pol and rev are in reverse and/or alternating orientations relative to one another; and wherein the cell is capable of producing a retroviral vector. 
     
     
         4 . A cell comprising nucleic acid sequences encoding:
 i) gag-pol;   ii) env;   iii) optionally the RNA genome of the retroviral vector; and   iv) optionally rev,   
       wherein the nucleic acid sequences are located at, at least, two different loci within the cell, further wherein at least two of the nucleic acid sequences are located at the same genetic locus in the genome of the cell; wherein at least one of the nucleic acid sequences encoding gag-pol or env is associated with at least one regulatory element; and wherein the cell is capable of producing a retroviral vector. 
     
     
         5 . A cell comprising nucleic acid sequences encoding:
 i) gag-pol;   ii) env;   iii) optionally the RNA genome of the retroviral vector; and   iv) rev,   
       wherein the nucleic acid sequences are located at, at least, two different loci within the cell, further wherein at least two of the nucleic acid sequences are located at the same genetic locus; wherein the nucleic acid sequences encoding rev and env are in reverse and/or alternating orientations relative to one another; and wherein the cell is capable of producing a retroviral vector. 
     
     
         6 . A modular construct comprising at least two nucleic acid sequences selected from nucleic acid sequences encoding:
 i) gag-pol;   ii) env;   iii) the RNA genome of the retroviral vector; and   iv) rev,   
       wherein the at least two of the nucleic acid sequences are in reverse and/or alternating orientations relative to one another; and wherein:
 a) when the modular construct comprises a nucleic acid sequence encoding gag-pol, said nucleic acid sequence encoding gag-pol is associated with at least one regulatory element; or 
 b) when the modular construct comprises a nucleic acid sequence encoding env, said nucleic acid sequence encoding env is associated with at least regulatory element; or 
 c) when the modular construct comprises nucleic acid sequences encoding gag-pol and env, said nucleic acid sequences encoding gag-pol and env are associated with at least one regulatory element. 
 
     
     
         7 . A modular construct comprising at least two nucleic acid sequences selected from nucleic acid sequences encoding:
 i) gag-pol;   ii) env;   iii) the RNA genome of the retroviral vector; and   iv) rev,   
       wherein the modular construct does not contain a sequence derived from a PAC, BAC, YAC, cosmid or fosmid; and wherein:
 a) when the modular construct comprises a nucleic acid sequence encoding gag-pol, said nucleic acid sequence encoding gag-pol is associated with at least one regulatory element; or 
 b) when the modular construct comprises a nucleic acid sequence encoding env, said nucleic acid sequence encoding env is associated with at least one regulatory element; or 
 c) when the modular construct comprises nucleic acid sequences encoding gag-pol and env, said nucleic acid sequences encoding gag-pol and env are associated with at least one regulatory element. 
 
     
     
         8 . A modular construct comprising nucleic acid sequences encoding env and rev, in reverse and/or alternating orientations relative to one another, optionally a nucleic acid sequence encoding gag-pol, and optionally the RNA genome of the retroviral vector. 
     
     
         9 . A modular construct comprising nucleic acid sequences encoding env and rev, in reverse and/or alternating orientations relative to one another, optionally a nucleic acid sequence encoding gag-pol, and optionally the RNA genome of the retroviral vector, wherein the modular construct does not contain a sequence derived from a PAC, BAC, YAC, cosmid or fosmid. 
     
     
         10 . A cell comprising at least one modular construct of  claim 6 , wherein the cell is capable of producing a retroviral vector. 
     
     
         11 . A method for generating a retroviral vector production cell, comprising the steps of:
 a) introducing at least one modular construct according to  claim 6  into a mammalian cell; and   b) optionally selecting for a mammalian cell which has the at least two nucleic acid sequences integrated within its genome;   thereby generating a retroviral vector production cell.   
     
     
         12 . The method according to  claim 11 , wherein integration of the at least two nucleic acid sequences is stable. 
     
     
         13 . The method according to  claim 11 , wherein integration of the at least two nucleic acid sequences is transient. 
     
     
         14 . A method for generating a retroviral vector production cell, comprising the steps of:
 a) introducing at least one modular construct according to  claim 7  into a mammalian cell; and   b) optionally selecting for a mammalian cell which has the at least two nucleic acid sequences integrated within its genome;   thereby generating a retroviral vector production cell.   
     
     
         15 . A method for generating a retroviral vector production cell, comprising the steps of:
 a) introducing at least one modular construct according to  claim 8  into a mammalian cell; and   b) optionally selecting for a mammalian cell which has the at least two nucleic acid sequences integrated within its genome;   thereby generating a retroviral vector production cell.   
     
     
         16 . A method for generating a retroviral vector production cell, comprising the steps of:
 a) introducing at least one modular construct according to  claim 9  into a mammalian cell; and   b) optionally selecting for a mammalian cell which has the at least two nucleic acid sequences integrated within its genome;   thereby generating a retroviral vector production cell.   
     
     
         17 . A method for producing a replication defective retroviral vector, comprising the steps of:
 a) introducing a modular construct according to  claim 6  into a mammalian cell;   b) selecting for a mammalian cell which has the at least two nucleic acid sequences integrated within its genome;   c) optionally introducing a nucleic acid vector which is different from the modular construct into the selected mammalian cell; and   d) further culturing the mammalian cell under conditions in which the replication defective retroviral vector is produced.   
     
     
         18 . A method for producing a replication defective retroviral vector, comprising the steps of:
 a) introducing a modular construct according to  claim 7  into a mammalian cell;   b) selecting for a mammalian cell which has the at least two nucleic acid sequences integrated within its genome;   c) optionally introducing a nucleic acid vector which is different from the modular construct into the selected mammalian cell; and   d) further culturing the mammalian cell under conditions in which the replication defective retroviral vector is produced.   
     
     
         19 . A method for producing a replication defective retroviral vector, comprising the steps of:
 a) introducing a modular construct according to  claim 8  into a mammalian cell;   b) selecting for a mammalian cell which has the at least two nucleic acid sequences integrated within its genome;   c) optionally introducing a nucleic acid vector which is different from the modular construct into the selected mammalian cell; and   d) further culturing the mammalian cell under conditions in which the replication defective retroviral vector is produced.   
     
     
         20 . A method for producing a replication defective retroviral vector, comprising the steps of:
 a) introducing a modular construct according to  claim 9  into a mammalian cell;   b) selecting for a mammalian cell which has the at least two nucleic acid sequences integrated within its genome;   c) optionally introducing a nucleic acid vector which is different from the modular construct into the selected mammalian cell; and   d) further culturing the mammalian cell under conditions in which the replication defective retroviral vector is produced.   
     
     
         21 . A method for producing a replication defective retroviral vector, comprising the steps of:
 a) introducing a modular construct according to  claim 6  into a mammalian cell;   b) optionally introducing a nucleic acid vector which is different from the modular construct into the mammalian cell; and   c) further culturing the mammalian cell under conditions in which the replication defective retroviral vector is produced.   
     
     
         22 . A method for producing a replication defective retroviral vector, comprising the steps of:
 a) introducing a modular construct according to  claim 7  into a mammalian cell;   b) optionally introducing a nucleic acid vector which is different from the modular construct into the mammalian cell; and   c) further culturing the mammalian cell under conditions in which the replication defective retroviral vector is produced.   
     
     
         23 . A method for producing a replication defective retroviral vector, comprising the steps of:
 a) introducing a modular construct according to  claim 8  into a mammalian cell;   b) optionally introducing a nucleic acid vector which is different from the modular construct into the mammalian cell; and   c) further culturing the mammalian cell under conditions in which the replication defective retroviral vector is produced.   
     
     
         24 . A method for producing a replication defective retroviral vector, comprising the steps of:
 a) introducing a modular construct according to  claim 9  into a mammalian cell;   b) optionally introducing a nucleic acid vector which is different from the modular construct into the mammalian cell; and   c) further culturing the mammalian cell under conditions in which the replication defective retroviral vector is produced.