In the late 1970s, it was predicted that gene therapy would be applied to humans within a decade. However, despite some success, gene therapy has still not become a routine practise in medicine. In this review, we will examine the problems, both experimental and clinical, associated with the use of viral material for transgenic insertion. We shall also discuss the development of viral vectors involving the most important vector types derived from retroviruses, adenoviruses, herpes simplex viruses and adeno‐associated viruses. This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009

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†These authors contributed equally to this study.Retroviral core proteins, Gag and envelope (Env) glycoproteins are expressed from distinct cellular areas and therefore need to encounter to assemble infectious particles. The intrinsic cell localisation properties of either viral component or their capacity to mutually interact determines the assembly of infectious particles. Here, we address how Env determinants and cellular sorting proteins allow the Env derived from gamma retroviruses, murine leukemia virus (MLV) and RD114, to travel to or from late endosomes (LE), which may represent the Env assembly site of retroviruses in some cells. The individual expression of MLV Env resulted in its accumulation in LE in contrast to RD114 Env that required the presence of gamma retroviral Gag proteins. To discriminate between intrinsic intracellular Env localisation and gamma retroviral Gag/Env interactions in influencing Env viral incorporation, we studied Env assembly on heterologous lentiviral particles on which they are passively recruited. We found that an acidic cluster present at the C-terminus of the RD114 Env cytoplasmic tail determines its sub-cellular localisation and retrograde transport. Mutation of this motif induced late endosomal concentration of the RD114 Env, correlating with increased viral incorporation and infectivity. Reciprocally, the reinforcement of a poorly functional acidic motif in the MLV Env resulted in a marked decrease of its late endosomal localisation, leading to weakly infectious lentiviral particles with low Env densities. Finally, through upregulation versus downregulation of its cellular expression, we show that phosphofurin acidic-cluster-sorting protein 1 (PACS-1) controls the function of the RD114 Env acidic cluster, assigning to this cellular effector a crucial role in modulation of Env assembly of some retroviruses.