Effect of nuclear localization and hydrodynamic delivery- induced cell division on phiC31 integrase activity

Phage φC31 integrase is a recombinase that can be expressed in mammalian cells to integrate plasmids carrying an attB sequence into the genome at specific pseudo attP locations. We demonstrate by immunofluoresence that wild-type φC31 integrase is cytoplasmic and that addition of a SV40 nuclear localization signal (NLS) localizes φC31 integrase to the nucleus. Unexpectedly, the NLS depressed integration efficiency in HeLa cells and provided no benefit when used to integrate the human Factor IX (hFIX) gene into mouse liver. Since breakdown of the nuclear membrane during mitosis could allow cytoplasmic integrase access to the chromosomes, we analyzed whether cell division was required for integration into liver cells in vivo. Hepatocytes were labeled with iododeoxyuridine to mark cells that underwent DNA replication during the week following hydrodynamic injection. Hydrodynamic delivery led to DNA replication in one-third of hepatocytes. Approximately 3 out of 4 cells having φC31 integrase-mediated stable hFIX expression did not undergo replication, indicating that cell division was not required for integrase function in liver. Therefore, although the bulk of φC31 integrase protein appears to be cytoplasmic in mammalian cells, integration can still occur in the nucleus, even without cell division. Keywords hydrodynamic delivery; liver; non-viral gene therapy; proliferation; nuclear localization signa