Nonviral gene therapy strives to eliminate cancer by correcting the molecular cause of the disease. Corrective DNA sequences, in the form of plasmids (pDNA), must localize to the cell nucleus to obtain transgene expression. This low probability event contributes to inefficient transgene expression following nonviral gene delivery. Previous studies using a bacteriophage (phage) display approach modified for the discovery of intracellular ligands yielded a panel of 7-mer peptides associated with the nuclei of MCF7 human mammary epithelial cells. The phage clone with the highest frequency of nuclear recovery displayed the QPSPSPT peptide. This study confirms the nuclear targeting capability of QPSPSPT and suggests the potential for QPSPSPT as a novel nonviral gene delivery ligand to modulate transgene expression in human breast cancer cells. The capacity for nuclear localization mediated by QPSPSPT was confirmed by image analysis of in vitro cell cultures exposed to fluorescently-labeled M13 phage displaying the heptapeptide. Control, wild-type M13 phage and phage displaying the QPSPSPT heptapeptide were labeled using an AlexaFluor 488 through conjugation to the pVIII coat proteins. Fluorescence microscopy revealed that 12.2% of MCF7 cell nuclei were associated with fluorescently labeled phage displaying the QPSPSPT peptide, but that wild-type M13 phage failed to localize to the cell nucleus. Quantitative analysis of confocal fluorescence micrographs enabled determination of intracellular and intranuclear phage localization. The number of phage per MCF7 nucleus was 3.3-fold greater for samples displaying the QPSPSPT peptide relative to the wild type M13 population. The increase in nuclear localization was mediated by both an increase in the amount of QPSPSPT-phage per cell (1.7-fold) and the intracellular translocation of QPSPSPT-phage to the nucleus (1.9-fold) as compared to the wild-type M13 phage. The equivalent spherical diameter of an M13 phage is approximately 40 nm, suggesting that the QPSPSPT heptapeptide has the capacity to mediate the cellular entry and nuclear localization of payloads consistent with the size of pDNA or formulated nonviral gene delivery vehicles. A commercial pDNA (pDualGC, Stratagene) was modified to enable the expression of fusion products consisting of a green fluorescent protein (GPF) bearing an N-terminus triple repeat of either the QPSPSPT or the positive control YSPTSPS. Transfection of these new constructs was performed independently in cultured MCF7 cells using Lipofectamine (Invitrogen). Six hours after transfection, the GFP-QPSPSPTx3 and GFP-YSPTSPSx3 fusion proteins appeared predominately in the cell nucleus in comparison with the cytosolic distribution of wild-type GFP. QPSPSPT has the capacity to preferentially localize proteins and phage to the cell nucleus, suggesting a potential role for this novel heptapeptide as a nonviral gene delivery adjuvant in breast cancer therapy. This work was funded by the BCRP-CDMRP under awards BC023276 and BC044010.
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6023.
Thirty-first San Antonio Breast Cancer Symposium Dec 10-14, 2008; San Antonio, TX