A new method of microcell-mediated transfer of human artificial chromosomes using a hemagglutinating virus of Japan envelope

概要

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Human artificial chromosomes (HACs) that segregate freely from the host chromosomes are potentially useful in therapeutic gene delivery for ensuring both safety and long-term gene expression. The polyethylene glycol (PEG)-based microcell-mediated chromosome transfer (MMCT) method has been recognized to be as the most suitable method for HAC transfer. However, the use of HAC vectors has been hampered by their low transfer efficiency and inability to achieve in vivo transfer. To circumvent these problems, we applied a hemagglutinating virus of Japan envelope (HVJ-E) vector system, a novel fusion and delivery system that has previously been shown to transfect plasmid DNA with greater efficiency and it has also been shown to be applicable to in vivo transfection into organs. A HAC vector carrying an EGFP transgene (21ΔqHAC-EGFP) was used to monitor the HAC transfer. The best results were obtained using the GenomONE-based fusion method combined with PHA-P ; the transfer efficiencies were then approximately three times and eight times higher than those obtained using the PEG-based MMCT method in HT1080 and hiMSC, respectively. Fluorescence in situ hybridization analysis showed 21ΔqHACEGFP to be present with a constant number of autonomous minichromosomes, as observed in the clones obtained by the PEG-based method. This HVJ-E vector system is therefore considered to be an improved method for HAC transfer.
財団法人染色体学会の論文