10 lentiviral vectors have indicated that they are able to efficiently replicate and express the transgene (Fig. 4). Targeted gene transfer and regulatable expression Various modifications have been employed in an effort to improve the targeting of viral vectors to specific cells and regulate transgene expression. Improved targeting has primarily been achieved by pseudotyping, altering the uptake of viral particles into specific cells by altering viral surface proteins/ envelope proteins to that of another viral subtype. Such retargeting of Ad vectors away from its primary receptor (CAR) to a tissue- or cell-specific receptors has been achieved by utilising different serotypes of adenovirus (Havenga et al. 2002). In addition, some rare HAd serotypes (Stone and Lieber 2006) and non- human Ads (Bangari and Mittal 2006) are now also being developed and investigated as alternate vectors for gene delivery. In addition to pseudotyping, targeting can be altered by specifically altering the viral capsid proteins by mutations, genetically mod- ifying the fibre and knob fibre proteins (Bakker et al. 2001; Nicklin et al. 2001). Such modifications can (1) increase the tropism of Ad vectors, making cells normally refractory to transduction (due to low CAR expression) accessible targets; (2) restrict transduction to the organ of interest enabling a lower dose to be used and (3) limit transgene transcription to certain cell types. Various studies have now demonstrated Cell Biol Toxicol (2010) 26:1–20 that the incorporation of additional RGD motifs on Ad fibre knobs can enhance the Ad transduction to a wide variety of cells (including endothelial cells, smooth muscle, fibroblasts, numerous tumour cell types and dendritic cells) that express low levels of CAR but high levels of integrins (Okada et al. 2001; Staba et al. 2000; Wickham et al. 1997). In addition, manipulation of such motifs may minimise the innate immune response by limiting vector interaction with the reticuloendothelial system, potentially avoiding the effects of an anti-adenoviral neutralising antibody response, and reduce the enhanced tropism for hepatocytes often displayed by Ad vectors. The latter is due to binding of coagulation factor IX, comple- ment component C4-binding protein and vitamin K- dependent blood coagulation factors (FVII, FIX, FX and protein C) to the Ad fibre knob or hexon protein which then acts as a link for virus uptake by hepatocytes through heparan sulfate proteoglycan or LDL receptor-related protein (Arnberg 2009; Parker et al. 2006; Waddington et al. 2008). Such retargeting will ultimately reduce systemic toxicity, especially following peripheral administration of Ad vectors. The targeting of lentiviral vectors has been modified in a similar way, utilising the envelope proteins of various retroviruses to enhance targeting to a particular cell type (Bouard et al. 2009 for review). Early versions of recombinant HIV vectors were of limited use due to their strict host cell tropism (CD4+ targets; Poznansky et al. 1991; Shimada et al. 1991). Pseudotyping with the vesicular stomatitis virus G Fig. 4 Non-integrating lentiviral vectors. Modification of lentiviral plasmids to contain an integrase negative helper plasmid have enabled the development of non-integrating lentiviral vectors. The viral genome does not integrate into the host genome but remains episomal. Transduction with EGFP- expressing virus demonstrates the efficiency of these vectors: a Integrating lenti-CMV-EGFP, b non-integrating CMV-EGFP and c non-integrating synapsin-EGFP