887. Reducing the Incidence of Replication Competent Adenovector by Engineering the Vector Genome

Replication-competent adenovirus (RCA) can arise through homologous recombination between the 293 cell chromosomal locus that contains a portion of the adenovirus DNA, and the vector genome. Two cross-over events are involved; one at the 5' end (|[sim]|350bp) and a second in the |[sim]|1000bp stretch that includes E1b, pIX and a portion of pIVa2. During scale up of the first generation adenoviral vector for clinical gene therapy trials, we observed that such recombinants arose in the production lots at a level close to the FDA recommended limit (fewer than 3 RCA in 1011 vector particles). In order to decrease the likelihood that RCA will contaminate large production lots, we decided to reorient a portion of the vector that is homologous with the Adenoviral sequences present in the 293 chromosome. We achieved this by inverting the protein IX gene and its upstream promoter region in E1b. Although we have not removed homologous bases from the vector, we hypothesize that disrupting the contiguous cis homology will reduce the probability of double cross over events that lead to RCA because only recombination within the untouched remaining 264bp of IVa2 can participate in the generation of replication-competent genomes. Tests for the frequency of RCA during serial passaging of the new vector indicate that it is at least 4 logs lower than the original design. Moreover, vector particle thermostability and transgene function were not adversely effected by this rearrangement. These data indicate that we have created a new vector with reduced incidence of RCA which in turn will facilitate the manufacturing process.