IMPROVED ADENOVIRUS PACKAGING CELL-LINES TO SUPPORT THE GROWTH OF REPLICATION-DEFECTIVE GENE-DELIVERY VECTORS

Adenovirus (Ad) vectors have been extensively used to deliver recombin ant genes to a great variety of cell types in vitro and in vivo, Ad-ba sed vectors are available that replace the Ad early region 1 (El) with recombinant foreign genes. The resultant El-deleted vectors can then be propagated on 293 cells, a human embryonal kidney cell line that co nstitutively expresses the EI genes. Unfortunately, infection of cells and tissues in vivo results in low-level expression of Ad early and l ate proteins (despite the absence of El activity) resulting in immune recognition of virally infected cells. The infected cells are subseque ntly eliminated, resulting in only a transient expression of foreign g enes in vivo, We hypothesize that a second-generation Ad vector with a deletion of viral genes necessary for Ad genome replication should bl ock viral DNA replication and decrease viral protein production, resul ting in a diminished immune response and extended duration of foreign gene expression in vivo, As a first step toward the generation of such a modified vector, we report the construction of cell lines that not only express the EI genes but also constitutively express the Ad serot ype 2 140-kDa DNA polymerase protein, one of three virally encoded pro teins essential for Ad genome replication. The Ad polymerase-expressin g cell lines support the replication and growth of H5ts36, an Ad with a temperature-sensitive mutation of the Ad polymerase protein. These p ackaging cell lines can be used to prepare Ad vectors deleted for the El and polymerase functions, which should facilitate development of vi ral vectors for gene therapy of human diseases.