We developed a novel method for delivery of human artificial chromosomes (HAC) into human cells using the Herpes Simplex Virus 1 (HSV-1) helper virus free system. This extremely efficient method has allowed gene expressing HACs for the first time to be established in a range of cell types including glioma, kidney and lung-derived cells.HACs are extrachromosomal molecules that behave as normal chromosomes in human cells and can be generated de novo in human cells following the introduction of vectors containing human alpha satellite (alphoid) DNA, the major sequence present at human centromeres. The HSV-1 technology allows the generation of highly infectious particles as amplicons for transduction of mammalian cells without viral propagation of the HSV-1 particles. HSV-1 has a large insert capacity (up to 150Kb) suitable for incorporating a long stretch of alphoid DNA, and infects a broad range of both dividing and non-dividing cells.HAC vectors containing 17 or 21 alpha satellite (alphoid) DNA, HSV elements, and genes for neomycin resistance (neo) and GFP were packaged into an HSV-1 capsid in African Green Monkey cells which generated high titre infectious HSV-1 HAC amplicons for transduction. Following infection of HSV-1 HAC amplicons in human HT1080 (fibrosarcoma), G16-9 (glioma), MRC5V2 (lung fibroblast), 293 (kidney epitheliod), BeFA (keratinocyte) and MRC5 (primary fibroblast), the efficiency of GFP expression after 24 hours was up to 10.000 times higher than standard HAC delivery methods. We successfully generated stable clones containing gene expressing HACs in HT1080, MRC5V2, 293 and G16-9. By including an HPRT minigene on the 21 alphoid HSV-1 HAC vector, we obtained functional HPRT HACs that complemented the HPRT deficiency in HT1080 cells. Studies on HAC stability revealed important differences between cell lines which have implications for HAC development in human cells. This work shows that the HSV-1 amplicon vector system is a significant advance for HAC delivery and the development of HAC gene expression systems in human cells.Molecular Therapy (2006) 13, S60–S60; doi: 10.1016/j.ymthe.2006.08.176 [ABSTRACT FROM AUTHOR]