Extrachromosomal DNA is becoming widely utilized as a gene therapy vector. Plasmid DNA offers multiple advantages over viral gene therapy vectors, including large packaging capacity, stability without integration and reduced toxicity. Furthermore, plasmid DNA can be delivered to many different tissues, using a variety of delivery techniques currently being developed. This review will discuss the advantages of extrachromosomal DNA as a gene therapy vector, highlighting recent advances and successes in its use in vivo.