Stocks of replication-deficient adenovirus

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 recombinant adenoviruses, wherein each recombinant adenovirus has a genome, the genome comprising:
 a nucleic acid encoding pIX gene product;   an expression cassette encoding a nonadenovirus gene product;   an adenoviral encapsidation signal; and   a 5′ adenoviral ITR or a derivative thereof and a 3′ adenoviral ITR or a derivative thereof;   wherein the recombinant adenovirus genome does not contain sequences of adenovirus E1A, E1B 21 kDa and E1B 55 kDa coding regions; and   wherein said stock of the recombinant adenoviruses is free of replication-competent adenovirus.   
     
     
         2 . The stock of  claim 1 , wherein the deletion in the at least part of the E1 region comprises a deletion of nucleotides corresponding nt. 459 to nt. 3510 of the adenovirus 5 genome. 
     
     
         3 . The stock of  claim 1 , wherein said recombinant adenovirus is capable of replication in a packaging cell line. 
     
     
         4 . The stock of  claim 3 , wherein said packaging cell line comprises a cell line deposited at the European Collection of Animal Cell Cultures under No. 96022940. 
     
     
         5 . The stock of  claim 1 , wherein the genome of said recombinant adenovirus further comprises other adenovirus sequences capable of allowing replication of said recombinant adenovirus in an E1-complementing cell. 
     
     
         6 . The stock of  claim 2 , wherein the genome of said recombinant adenovirus further comprises other adenovirus sequences capable of allowing replication of said recombinant adenovirus in an E1-complementing cell. 
     
     
         7 . The stock of  claim 4 , wherein the stock of recombinant adenoviruses is capable of being produced in the packaging cell line on a large scale. 
     
     
         8 . A stock of recombinant adenoviruses, each recombinant adenovirus thereof having a genome, the genome comprising:
 a nucleic acid encoding pIX gene product;   an expression cassette encoding a nonadenovirus gene product;   an adenoviral encapsidation signal; and   a 5′ adenoviral ITR or a derivative thereof and a 3′ adenoviral ITR or a derivative thereof;   wherein the genome does not contain sequences of adenovirus E1A, E1B 21 kDa and E1B 55 kDa coding regions and further has a deletion of one or more of adenovirus E2A and adenovirus E3; and   wherein said stock is free of replication-competent adenovirus.