Fig. 3

Gene transfer using lentiviral vectors. Third-generation lentiviral vectors are produced by transfecting producer cells with the four packaging plasmids, which will initiate transcription of Gag and GagPol polyprotein precursors, the envelope glycoprotein (e.g. VSV-G), Rev and the transfer vector carrying the (trans)gene of interest (GOI) to be inserted into the target cells. Nascent lentiviral particles are packaged together with an RNA dimer encoding the transgene flanked by viral cis-elements required for RNA packaging and reverse transcription. Budding of lentiviral particles results in immature particles, which are then matured in a process involving cleavage of the Gag and GagPol polyproteins as well as formation of the viral core. Uptake into target cells is achieved through receptor-mediated endocytosis, following which the viral core is released into the cytoplasm. Reverse transcription of the transfer vector single-stranded RNA then occurs, resulting in double-stranded DNA, which is then transported into the nucleus and integrated into the genome of the target cell