C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya

In contrast to the standard nuclear expression systems that require access to the host-cell nuclear transcriptional machinery, the C3P3-G1 system has been devised as an autonomous cytoplasmic expression system. Therefore, once expression of the C3P3 enzyme has been primed, it becomes independent of the passive diffusion of the input DNA through the nuclear pores. The diameter of their main channel, which is smaller than the classical diameter of plasmid DNA, explains their limited diffusion to the nucleus and possibly the limited efficacy of transient transfection (78) . For example, only 1-10% of the plasmids transfected to cultured cells can effectively reach their nuclei as assessed by quantitative RT-PCR, Southern blot analysis, or electron microscopy (79, 80) . Hence, the cytoplasmic processivity of the C3P3 system appears appealing for transient non-viral gene therapy, an attractive approach to deliver therapeutic Figure 11 . Kinetics of expression of secreted proteins by CHO-K1 cells. Titers of (A) the secreted human erythropoietin (hEPO), (B) human granulocyte colony-stimulating factor isoform-b (hG-CSF) and, (C) secreted/cytoplasmic mouse alpha-fetoprotein (mAFP) released in serumfree culture medium of CHO-K1 cells were monitored by ELISA.