Several viruses have been engineered for gene therapy applications, and the specific properties of each viral vector have been exploited to target a variety of inherited and acquired diseases. Preclinical and clinical studies demonstrated that viral vectors are highly versatile tools capable of efficient transfer of foreign genetic information into almost all cell types and tissues. Gene therapy applications depend on vector characteristics, such as host range, cell- or tissue-specific targeting, genome integration, efficiency and duration of transgene expression, packaging capacity, and suitability for scale-up production. This review discusses the advances in the development of viral vectors, with particular emphasis on how knowledge of virus biology has been exploited to design a variety of vectors with improved safety characteristics and efficiency, potentially suitable for a large number of gene therapy applications.