The discovery of autonomous replicating sequences (ARSs) in Saccharomyces cerevisiae in 1979 was considered a milestone in unraveling the regulation of replication in eukaryotic cells. However, shortly afterwards it became obvious that in Saccharomyces pombe and all other higher organisms ARSs were not sufficient to initiate independent replication. Understanding the mechanisms of replication is a major challenge in modern cell biology and is also a prerequisite to developing application-oriented autonomous replicons for gene therapeutic treatments. This review will focus on the development of non-viral episomal vectors, their use in gene therapeutic applications and our current knowledge about their epigenetic regulation. 