The clinical application of gene therapy has become a reality with the treatment of patients with X-linked SCID (SCID-X1) using a modified retrovirus. This success has been tempered by the toxicity of the vector used in this trial, which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors, which remain episomal and are therefore less genotoxic, is currently an area of active research. Notable recent developments include the application of modified lentiviral vectors, which stably express transgenes without the risk of integration; plasmid vectors, which exist episomally and are persistently expressed in the livers of mice; and the generation of replicating artificial chromosomes containing genomic loci. In addition, knowledge of the molecular mechanisms of nuclear retention and replication of the transgene is improving and will facilitate further developments in the use of episomal DNA for the genetic modification of cells. This review describes the development and application of gene therapy vectors, with a focus on those that are specifically designed to avoid integration and exist episomally.