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 . An isolated adenovirus packaging cell comprising in its genome: 
 a first nucleic acid sequence encoding adenovirus E1A and E1B gene products but lacking a nucleic acid sequence encoding adenovirus pIX.    
     
     
         2 . (canceled)  
     
     
         3 . The isolated adenovirus packaging cell of  claim 1 , wherein said first nucleic acid sequence comprises nucleotides 459-3510 of the human adenovirus 5 genome.  
     
     
         4 . The isolated adenovirus packaging cell of  claim 1 , wherein the isolated adenovirus packaging cell is of a retina cell origin.  
     
     
         5 . The isolated adenovirus packaging cell of  claim 1 , wherein the isolated adenovirus packaging cell is of a primary cell origin.  
     
     
         6 . The isolated adenovirus packaging cell of  claim 1 , wherein the isolated adenovirus packaging cell is of an embryonal cell origin.  
     
     
         7 . The isolated adenovirus packaging cell of  claim 1 , wherein the isolated adenovirus packaging cell is a human cell.  
     
     
         8 . (canceled)  
     
     
         9 . The isolated adenovirus packaging cell of  claim 1 , wherein the isolated adenovirus packaging cell is a PER.C6 cell as deposited under no. 96022940 at the European Collection of Animal Cell Cultures.  
     
     
         10 . The isolated adenovirus packaging cell of  claim 1 , further comprising a nucleic acid sequence encoding an adenovirus E2A gene product.  
     
     
         11 . The isolated adenovirus packaging cell of  claim 10 , wherein the adenovirus E2A gene product includes a temperature sensitive 125 mutation.  
     
     
         12 - 15 . (canceled)  
     
     
         16 . The isolated adenovirus packaging cell of  claim 1 , wherein the isolated adenovirus packaging cell is of human embryonic retinoblast (HER) origin.  
     
     
         17 - 20 . (canceled)  
     
     
         21 . The isolated adenovirus packaging cell of  claim 1 , further comprising a recombinant expression vector derived from a human adenovirus genome, wherein said expression vector comprises an adenovirus gene encoding a pIX protein and further wherein said expression vector lacks nucleic acid sequences that overlap with said first nucleic acid sequence.  
     
     
         22 . The isolated adenovirus packaging cell of  claim 10 , wherein the nucleic acid sequence encoding an adenovirus E2A protein is operatively linked to an E1A-independent transcription initiation region.  
     
     
         23 . The isolated adenovirus packaging cell of  claim 21 , wherein the recombinant expression vector is IG.Ad.MLPI.TK shown in  FIG. 12 .  
     
     
         24 . The isolated adenovirus packaging cell of  claim 21 , wherein the recombinant expression vector is derived from a human adenovirus 5 genome from which nucleotides 459-3510 have been deleted.  
     
     
         25 - 28 . (canceled)