MOLECULAR CHARACTERIZATION OF REPLICATION-COMPETENT VARIANTS OF ADENOVIRUS VECTORS AND GENOME MODIFICATIONS TO PREVENT THEIR OCCURRENCE

Adenovirus (Ad) vectors for gene therapy are made replication defectiv e by deletion of E1 region genes. For isolation, propagation, and larg e-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical s tudies are produced in the 293 cell, a human embryonic kidney cell lin e expressing E1 functions from an integrated segment of the left end o f the Ad type 5 (Ad5) genome. Replication-competent vector variants th at have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells, These replication-competent variants p resumably result from recombination between vector and 293 cell Ad5 se quences, We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All s uch variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E 1 sequences have become incorporated into the viral genome by legitima te recombination events, A map of Ad5 sequences within the 293 cell ge nome developed in parallel is consistent with the proposed recombinati on events, To provide a convenient vector production system that circu mvents the generation of replication-competent variants, we have modif ied the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequen ces is greatly reduced, Twelve serial passages of an Ad2 vector lackin g the protein IX gene were carried out without generating replication- competent variants, In the course of producing and testing more than 3 0 large-scale preparations of vectors lacking the protein IX gene or h aving a rearranged protein IX gene, only three examples of replication -competent variants were observed, Use of these genome modifications a llows use of conventional 293 cells for production of large-scale prep arations of Ad-based vectors lacking replication-competent variants.