Packaging systems for human recombinant adenovirus to be used in gene therapy

Abstract

Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector are suited for large scale production of recombinant adenoviruses. A system for use with the invention produces replication-defective adenovirus. The system includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, has at least one functional encapsidation signal and at least one functional Inverted Terminal Repeat, and lacks overlapping sequences with the nucleic acid of the cell. Otherwise, the overlapping sequences would enable homologous recombination leading to replication competent adenovirus in the primary cell into which the isolated recombinant nucleic acid molecule is to be transferred.

1 . A stock of a replication deficient adenoviral gene transfer vector, said replication deficient adenoviral gene transfer vector comprising: 
 an expression cassette encoding a foreign gene product;    an adenoviral encapsidation signal; and    at least one copy of at least a functional part of an adenoviral ITR or a derivative thereof, wherein said stock is free of replication competent adenovirus.    
     
     
         2 . The stock of  claim 1 , wherein said replication deficient adenoviral gene transfer vector comprises a deletion in at least a part of the E1 region of the adenoviral genome.  
     
     
         3 . The stock of  claim 2 , wherein said part of the E1 region comprises the E1A region.  
     
     
         4 . The stock of  claim 2 , wherein said part of the E1 region comprises nt. 459 to nt. 3510 of the adenoviral genome.  
     
     
         5 . The stock of  claim 2 , wherein said replication deficient adenoviral gene transfer vector further comprises a deletion of the E2A region of the adenoviral genome.  
     
     
         6 . The stock of  claim 2 , wherein said replication deficient adenoviral gene transfer vector further comprises genetic information encoding a pIX gene product and a pIX promoter.  
     
     
         7 . The stock of  claim 2 , wherein said replication deficient adenoviral gene transfer vector further comprises at least one deficiency in at least one additional part of the adenoviral genome.  
     
     
         8 . The stock of  claim 2 , where said replication deficient adenoviral gene transfer vector further comprises at least one deficiency in at least one additional part of the adenoviral genome necessary for viral replication.  
     
     
         9 . The stock of  claim 4 , wherein said replication deficient adenoviral gene transfer vector replicates in a packaging cell line.  
     
     
         10 . The stock of  claim 9 , wherein said packaging cell line comprises the PER.C6™ cell line.  
     
     
         11 . A stock of a replication deficient adenoviral gene transfer vector, said vector comprising: 
 an expression cassette encoding a foreign gene product;    an encapsidation signal comprising a portion of the viral genome required for encapsidation of the genome into virus particles; and    at least one copy of at least a functional part of an adenoviral ITR or a derivative thereof, wherein said stock is free of replication competent adenovirus.    
     
     
         12 . The stock of  claim 11 , wherein said replication deficient adenoviral gene transfer vector has a deletion in at least a part of the E1 region of the adenoviral genome.  
     
     
         13 . The stock of  claim 12 , wherein said part of the E1 region is the E1A region.  
     
     
         14 . The stock of  claim 12 , wherein said part of the E1 region comprises nt. 459 to nt. 3510 of the adenoviral genome.  
     
     
         15 . The stock of  claim 12 , wherein said replication deficient adenoviral gene transfer vector further comprises a deletion of the E2A region of the adenoviral genome.  
     
     
         16 . The stock of  claim 12 , wherein said replication deficient adenoviral gene transfer vector further comprises genetic information encoding a pIX gene product and a pIX promoter.  
     
     
         17 . The stock of  claim 12 , wherein said replication deficient adenoviral gene transfer vector further comprises at least one deficiency in at least one additional part of the adenoviral genome.  
     
     
         18 . The stock of  claim 12 , where said replication deficient adenoviral gene transfer vector further comprises at least one deficiency in at least one additional part of the adenoviral genome necessary for viral replication.  
     
     
         19 . The stock of  claim 14 , wherein said replication deficient adenoviral gene transfer vector replicates in a packaging cell line.  
     
     
         20 . The stock of  claim 19 , wherein said packaging cell line comprises the PER.C6™ cell line.