Transient transfection method for retroviral production

Abstract

The invention relates to bacterial artificial chromosomes (BAC) comprising retroviral nucleic acid sequences encoding: gag and pol proteins, and an env protein or a functional substitute thereof, wherein each of the retroviral nucleic acid sequences are arranged as individual expression constructs within the BAC. The invention also relates to uses and methods of transient transfection using said BAC.

1 . A nucleic acid vector selected from: a yeast artificial chromosome (YAC), a P1-derived artificial chromosome (PAC), a fosmid, or a cosmid, characterized in that said vector comprises retroviral nucleic acid sequences encoding: gag and pol proteins, and an env protein or a functional substitute thereof, wherein each of the retroviral nucleic acid sequences are arranged as individual expression constructs within the vector. 
     
     
         2 . The nucleic acid vector of  claim 1 , which additionally comprises nucleic acid sequences which encode the RNA genome of a retroviral vector particle. 
     
     
         3 . The nucleic acid vector of  claim 1 , which additionally comprises the auxiliary gene rev or an analogous gene thereto or a functionally analogous system. 
     
     
         4 . The nucleic acid vector of  claim 1 , wherein the retroviral nucleic acid sequences are obtained from a retrovirus selected from lentivirus, alpha-retrovirus, gammaretrovirus or foamy-retrovirus. 
     
     
         5 . The nucleic acid vector of  claim 4 , wherein the retroviral nucleic acid sequences are obtained from a lentivirus selected from the group consisting of HIV-1, HIV-2, SIV, FIV, EIAV and Visna. 
     
     
         6 . The nucleic acid vector of  claim 5 , wherein the retroviral nucleic acid sequences are obtained from HIV-1. 
     
     
         7 . The nucleic acid vector of  claim 1 , wherein the env protein or a functional substitute thereof is obtained from Vesicular stomatitis virus. 
     
     
         8 . The nucleic acid vector of  claim 1 , which additionally comprises a transcription regulation element. 
     
     
         9 . The nucleic acid vector of  claim 8 , wherein the transcription regulation element is a CMV promoter. 
     
     
         10 . The nucleic acid vector of  claim 1 , which additionally comprises an insulator. 
     
     
         11 . The nucleic acid vector of  claim 10 , wherein an insulator is present between each of the retroviral nucleic acid sequences. 
     
     
         12 . The nucleic acid vector of  claim 1 , which additionally comprises one or more transgenes. 
     
     
         13 . The nucleic acid vector of  claim 1 , which additionally comprises an Internal Ribosome Entry Site (IRES). 
     
     
         14 . The nucleic acid vector of  claim 1 , which additionally comprises a polyA signal. 
     
     
         15 . The nucleic acid vector of  claim 1 , which additionally comprises an intron sequence. 
     
     
         16 . The nucleic acid vector of  claim 1 , which additionally comprises a Multiple Cloning Site (MCS). 
     
     
         17 . A method of producing a replication defective retroviral vector particle, comprising:
 (a) introducing into a culture of mammalian host cells, a nucleic acid vector selected from: a yeast artificial chromosome (YAC), a P1-derived artificial chromosome (PAC), a fosmid, or a cosmid, characterized in that said vector comprises retroviral nucleic acid sequences encoding: gag and pol proteins, and an env protein or a functional substitute thereof wherein each of the retroviral nucleic acid sequences are arranged as individual expression constructs within the vector; and   (b) culturing the mammalian host cells under conditions in which the replication defective retroviral vector particle is produced.   
     
     
         18 . The method of  claim 17 , wherein the mammalian host cell is a HEK 293 cell. 
     
     
         19 . The method of  claim 17 , wherein introduction step (a) is performed using lipofection, electroporation or a chemical-based transfection method, such as calcium phosphate treatment. 
     
     
         20 . The method of  claim 17 , wherein culturing step (b) is performed by incubating the mammalian host cell under humidified conditions. 
     
     
         21 . The method of  claim 17 , additionally comprising isolating the replication defective retroviral vector particle. 
     
     
         22 . The method of  claim 21 , wherein the isolating is performed by using a filter, such as a low-protein binding membrane. 
     
     
         23 . The method of  claim 21 , wherein the replication defective retroviral vector particles are isolated no longer than 72 hours after introduction step (a). 
     
     
         24 . The method of  claim 23 , wherein the replication defective retroviral particles are isolated between 48 and 72 hours after introduction step (a).