Production of Lentiviral Vectors

Abstract

A high-volume gene therapy vector manufacturing process which produces a recombinant gene therapy vector which is able to transform host cells even when they are not dividing.

We claim: 
     
         1 . A method comprising:
 a. Transducing a mammalian cell with a baculovirus to make a transduced mammalian producer cell; and then   b. Culturing said transduced mammalian producer cell in culture media, and harvesting from said transduced mammalian producer cell and/or culture media a second virus having a therapeutic transgene.   
     
     
         2 . The method of  claim 1 , wherein said harvesting comprises harvesting at about 144 hours after said transduction. 
     
     
         3 . The method of  claim 1 , wherein said second virus is derived from a virus which in its wild state has a single-stranded genome. 
     
     
         4 . The method of  claim 1 , wherein said second virus is able to transduce a human cell which is not actively dividing. 
     
     
         5 . The method of  claim 1 , wherein said second virus is replication deficient. 
     
     
         6 . The method of  claim 1 , wherein said second virus can integrate in host genome at specific site and can produce stable long-term expression. 
     
     
         7 . The method of  claim 1 , wherein said second virus is non-immunogenic. 
     
     
         8 . A recombinant baculovirus having at least one nucleic acid sequence coding for a second virus derived from a virus which in its wild state has a single stranded genome, said second virus being replication-deficient and having a therapeutic transgene. 
     
     
         9 . The recombinant baculovirus of  claim 8 , wherein said second virus is able to transduce a human cell which is not actively dividing. 
     
     
         10 . The recombinant baculovirus of  claim 8 , wherein said second virus can integrate in the human genome and can produce stable long-term expression in a transduced human cell. 
     
     
         11 . A method comprising:
 a. Obtaining the recombinant baculovirus of  claim 8 ; and then   b. Transducing a mammalian producer cell with said recombinant baculovirus to make a transduced mammalian producer cell; and then   c. Culturing said transduced mammalian producer cell in culture media, and harvesting said second virus from said transduced mammalian producer cell and/or culture media.   
     
     
         12 . A viral vector able to transfect a human cell which is not actively dividing, said viral vector produced by a mammalian producer cell transduced with a recombinant baculovirus. 
     
     
         13 . The viral vector of  claim 12 , wherein said viral vector has a therapeutic transgene. 
     
     
         14 . The viral vector of  claim 12 , said viral vector comprises virus derived from a virus which in its wild state has a single stranded genome. 
     
     
         15 . A method comprising:
 a. Obtaining the viral vector of  claim 12 ; and   b. Transducing a human patient's cells with said viral vector.   
     
     
         16 . A method comprising:
 a. Obtaining the viral vector of  claim 13 ; and   b. Transducing a human patient's cells with said viral vector.   
     
     
         17 . A method comprising:
 a. Obtaining the viral vector of  claim 14 ; and   b. Transducing a human patient's cells with said viral vector.