775. An Novel Adenoviral E1 Complementary Cell for Eliminating Replication Competent Adenovirus (RCA) during Viral Production|[ast]|

Production of recombinant adenoviruses from 293 cells and other recently developed adenoviral E1-complementary cell lines have the potential to produce low levels of replication competent adenoviruses (RCA) derived from cellular DNA recombination. In most E1 complementary cells, varying lengths of the adenoviral E1A and E1B coding regions and their transcriptional regulatory structures have been used to generate the cell lines. A 3Kb fragment of the viral genome containing both the E1A and E1B coding regions (nts 560 to 3510 of adenovirus 5) is essential in developing such cell lines. This leaves open the possibility for cellular-derived E1 DNA to undergo homologous recombination with both E1-deleted replication defective and E1-modified replication competent adenoviral vectors. In order to mitigate the potential for cell-derived homologous recombination during adenovirus production, we applied a different strategy to generate E1-complementary cells. The coding sequences for adenovirus E1A and E1B were cloned into separate retroviral vectors derived from Moloney Murine Leukemia Virus (MMLV). E1A or E1B expressing transient retroviral supernatants were separately prepared from these vectors and used to co-infect cells. The A549 human lung carcinoma cell line was co-infected by the supernatants containing the MMLV-E1A and MMLV-E1B vectors. Limiting dilution and functional screens led to the isolation of adenoviral E1-complementary cell lines. These cells support the replication of E1-defective adenoviral vectors and express the E1A and E1B gene products as demonstrated by Western blot. Of note, instead of a continuous strand of adenoviral DNA, the E1A and E1B genes are separately integrated as two smaller DNA fragments. This arrangement is designed to minimize the chance of RCA generation derived from the recombination of viral DNA with cellular DNA. In addition to supporting the replication of E1-defective adenoviral vectors, further characterization of these cell lines demonstrated that they complement the replication of E1-modified replicating adenoviral vectors in which the E1 genes are under the control of tissue-specific, transcriptional regulatory elements. Comparisons of 293, PerC6, and the A549-derived E1-complementary cell lines suggest equivalent viral production. This complementation ability was stable over 10 weeks in culture and multiple passages of the cell lines. Preliminary RCA assessment suggests that these cells are superior to 293 cells and other cells in reducing the production of RCA.