Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. There is increasing agreement that the ideal vector for gene therapy should be completely based on chromosomal elements and behave as an independent functional unit after integration into the genome or when retained as an episome. In this review we will first discuss the chromosomal elements, such as enhancers, locus control regions, boundary elements, insulators and scaffold- or matrix-attachment regions, involved in the hierarchic regulation of mammalian gene expression and replication. These elements have been used to design vectors that behave as artificial domains when integrating into the genome. We then discuss recent progress in the use of mammalian artificial chromosomes and small circular non-viral vectors for their use as expression systems in mammalian cells.