Assaying extrachromosomal gene therapy vectors that carry replication/persistence elements.

Persistence in the cell is a desirable property for most gene therapy vectors. For extrachromosomal vectors, persistence is limited in most cell types. To address this problem, we have developed vectors with the ability to replicate and be retained in the nucleus. These properties are conferred by specific elements present on the vectors and derived from genomic DNA and from Epstein-Barr virus. In order to begin evaluation of these vectors for use in gene therapy, we developed and present here two assays that measure the persistence of vector DNA in tissue culture cells under rapidly dividing and slowly dividing conditions. Our results indicate that inclusion of DNA replication and nuclear retention elements on a vector increases persistence of vector DNA in slowly dividing cells by at least 500%. Further improvement of the system is discussed.