Cell Biol Toxicol (2010) 26:1–20 genes of HIV-1 (Delenda 2004; Kim et al. 1998; Zufferey et al. 1997). Third-generation lentiviral vectors see the replacement of the tat gene with powerful constitutive promoters, such as the immedi- ate early cytomegaly virus (CMV) promoter (Dull et al. 1998; Kim et al. 1998), increasing the bio-safety of these vectors. These minimal vector systems have the essential viral components segregated onto three or four separate plasmids with minimal sequence over- lap. An additional safety concern of using lentiviral- based vector systems is the possibility of insertional activation of an oncogene following random integra- tion of the vector provirus into the host genome. This problem has been addressed by the development of self-inactivating lentiviral vectors (Miyoshi et al. 1998; Xu et al. 2001). These vectors contain deletions in the U3 region of the 3′ LTR, which is transferred to the promoter in the 5′ LTR after reverse transcription and eliminates transcriptional activation of surround- ing host genes (Logan et al. 2004). Thus, recombinant lentiviral vectors are now constructed using only three HIV-1 genes: gag, pol and rev. The cloning vectors, into which genes of interest are placed, are devoid of all viral sequences apart from essential cis-acting sequences, including the LTRs and the packaging signal. The rev responsive element (RRE) is also included to ensure efficient nuclear export of the full- length viral RNA genome. Despite advances addressing safety concerns of using lentiviral vectors, there are still problems regarding limited transgene capacity, difficulties in constructing stable producer cell lines due to viral gene toxicity, and thus the inability to produce high- titre stocks. The reintroduction of other viral sequen- ces, such as the central polypurine tract (cPPT) and the central termination sequence from the HIV pol gene, improves gene expression (Barry et al. 2001; De Rijck et al. 2005; Logan et al. 2004). These sequences (regardless of their position within the viral vector; De Rijck et al. 2005) improve translocation of the pre-integration complex, resulting in increased nuclear transduction efficiency and production of higher viral titres. Incorporation of a posttranscrip- tional regulatory element into lentiviral vectors increases nuclear RNA export, stabilises the viral vector mRNA and hence increases translation and transgene expression (Zufferey et al. 1999). The majority of lentiviral vector development has focused on primate lentivirus-based systems, such as 9 HIV-1. However, other gene transfer systems based on non-primate lentiviruses, such as the feline immunodeficiency virus (FIV; Curran et al. 2000; Poeschla et al. 1998) and the equine infectious anaemia virus (EIAV; Olsen 1998), have also been developed. Since their initial development, these systems have been optimised, producing minimal cassettes that include additional regulatory elements (similar to those used in HIV-1-based systems; Johnston et al. 1999; Saenz and Poeschla 2004) and have been pseudotyped with alternative envelope glycoproteins (Mazarakis et al. 2001; Sinn et al. 2005; Song et al. 2003;Wong et al. 2004). These non- primate-based systems can successfully transduce dividing and non-dividing cells (Mitrophanous et al. 1999) and can mediate neuronal gene expression in vivo (Azzouz et al. 2004; Azzouz and Mazarakis 2004; Wong et al. 2005). Such systems are thought to be inherently safer, as humans are not the host of the wild-type virus, although early EIAV systems were limited by the instability of vector-derived RNA transcripts and silencing of the EIAV over time (O’Rourke et al. 2002). Development of these systems has eliminated such problems, and many studies show that EIAV vectors can now transduce human cells with similar efficiency to HIV-1. Gene therapy trials have highlighted concerns regarding the propensity for lentiviral vectors (includ- ing HIV-1) to integrate within transcriptionally active regions known as euchromatin. Genomic integration of lentiviral vector genome facilitates long-term expression as the viral sequence is replicated during mitosis. This desirable trait can also be a problem, especially when the insertion site is in close proximity to a proto-oncogene (Somia and Verma 2000). In recent clinical gene therapy trials for X-linked severe combined immunodeficiency, five children developed leukaemia as a direct consequence of insertional mutagenesis (Staal et al. 2008). In two cases, retrovirus integration was in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2 (Hacein-Bey- Abina et al. 2003). The development of non- integrating lentiviral vectors, where the viral genome remains episomal, may address such problems. Although such vectors offer a limited expression window in mitotic cells, good expression is produced in post-mitotic cells, such as neurons (Yanez-Munoz et al. 2006). Initial studies using non-integrating