Nonviral gene administration by means of the Epstein-Barr virus (EBV)-based episomal vectors and it application to gene therapy and regenerative medicine

摘要

Genetic transfection is a fundamental technology required for analysis and control of cells and tissues. The efficiency of gene transfection is drastically improved by using Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene and oriP, which are derived from EBV genome, as components of plasmid vectors to be combined with various non-viral gene transfer vehicles. By means of the multiscale manipulation procedures, we analyzed intracellular distribution of EBNA1 and oriP sequence after transfected the EBNAl/oriP-bearing plasmid vectors (EBV-based episomal vectors) after transfected into mammalian cells, and estimated its implication to the high rate transfection of the EBV-based episomal vectors. Because the EBNA1 plays pleiotrophic roles in transfected cells, we also evaluated contribution of each function of the molecule to the transfection and expression efficiency of the vectors. Beside these studies on basic aspects of the EBNAl/oriP system, we assessed possible application of the EBV-based episomal vectors to regenerative medicine and gene therapy. For example, in vivo activities of various cytokines were tested in normal as well as diseased animals by transfecting them in vivo with the EBV-based episomal vectors carrying expression units for the cytokine genes. Thus, systemic administration of the interleukin-27 (IL-27) gene into the mice that had been transplanted with squamous cell carcinoma resulted in significant suppression of the growth of the tumor, which was mediated by the cooperation of the tumor-specific IgG antibody that was induced by IL-27 and the natural killer (NK) cells of which cytotoxic activity was also enhanced by the cytokine. IL-28 gene also suppressed the tumor in the similar experimental setting using the squamous cell carcinoma, but unlike the mechanism of IL-27-mediated anti-tumor effect, the tumor inhibition resulted from IL-28 gene transfer may be mediated by induction of the cytotoxic T lymphocytes, while coadministration of a chemotherapeutic agent significantly enhanced the IL-28-mediated tumor suppression. There findings may propose novel gene therapy and immunotherapy procedures to concur malignancies, while the highly efficient and long-term persistent exogenous gene expression accomplished by the EBV-based episomal vector may also provide a powerful means for stem cell study and regenerative medicine for human diseases.