6 of gene transfer to the nervous system (Akli et al. 1993; Davidson et al. 1993). Wild-type Ads can only incorporate an additional 2 kb of foreign DNA without significant effects upon the stability of infectivity (Russell 2000). Removal of viral-encoded genes allows the incorporation of larger genetic sequences. First-generation Ad vectors were produced by removing the E1 and E3 viral cassettes, making way for approximately 8 kb of foreign DNA (Bett et al. 1994; Verma and Weitzman 2005). The removal of the E1 gene renders the virus extremely attenuated in its ability to replicate, and it is therefore necessary to propagate these viruses in a helper cell line that supplies the E1 gene in trans, such as the human embryonic kidney 293 (HEK293) cell line (Graham et al. 1977). The most commonly used methods to produce first-generation recombinant Ads rely on either homologous recombination in mammalian cells (Bett et al. 1994), homologous recombination in bacteria (He et al. 1998) or in vitro ligation (Mizuguchi and Kay 1998). Following homologous recombination in HEK293 cells, viral particles are screened for those expressing the inserted transgene by plaque purification. Further modifications to this method, including alterations made to the packaging cell lines, have reduced the need for this step and increased the efficiency of adenoviral production protocols (Davidson et al. 2000). Viral particles are released from HEK293 cells by sonication, separated by a CsCl gradient spin and purified by dialysis before storage. The high transduction efficiency of Ad vectors combined with their relative ease of prepara- tion and purification has led to their extensive use as gene transfer vectors (Hermens and Verhaagen 1998; Verma and Weitzman 2005). Initial in vivo studies conducted with first-generation Ad vectors revealed several limitations, including the initiation of significant systemic immune and inflamma- tory responses. This reduced the duration of transgene expression and limited safety and efficacy when used in vivo (Byrnes et al. 1995; Muruve 2004; Wilson 1996). Furthermore, under certain circumstances, cellular factors can transactivate viral genes that have E1A- responsive promoters and facilitate low-level DNA replication and the production of late structural anti- gens. Such problems are highly apparent when first- generation Ad vectors are injected into peripheral organs (Spergel et al. 1992; Spergel and Chen-Kiang 1991). Transgene expression is generally undetectable Cell Biol Toxicol (2010) 26:1–20 after 14 days due to activation of CD8+ T cells that subsequently eliminate the infected cell and induce a humoral response against antigens contained in the viral capsid (Yang et al. 1994a, 1995). In addition, significant toxicity, both in vitro and in vivo, has been reported when first-generation Ad vectors are used to infect cells at higher doses (or multiplicities of infection). To overcome such problems, extensive develop- ments in Ad vector biology and expression cassette design have been made. Second-generation vectors were developed to address the problems of immune recognition, containing deletions in the E2a and E2b genes that encode viral DNA binding protein and DNA polymerase, respectively (Engelhardt et al. 1994; Morral et al. 1997; Yang et al. 1994b). Ad vectors deleted of E4 genes have also been investigated (Dedieu et al. 1997). The resulting recombinant viral vectors can incorporate larger transgenes (up to 10 kbp), are less cytotoxic and systemically immunogenic, but are unable to produce high viral titres or mediate stable transgene expression. A significant advance in Ad vector technology has been the development of helper- dependent (Hd) or ‘gutless’ Ad vectors that are deleted of all Ad gene sequences except those required (in cis) for replication and packaging (Palmer and Ng 2005). A replication incompetent (helper) virus is therefore required to supply all of the necessary Ad functions in trans and is later separated from the recombinant virus (Parks et al. 1996). Consequently, the production of Hd-Ads is more complicated than earlier genera- tions, with problems in the longevity and levels of expression (Schiedner et al. 2002) as well as helper virus contamination (Alba et al. 2005). Due to the large amount of viral DNA removed, the cloning capacity of the vector is increased and the host adaptive response is minimised. This improves the efficacy and duration of gene transfer in vivo (Muruve 2004; Parks et al. 1996), and long-term expression in immunocompetent animals, including efficient delivery to the CNS (Cregan et al. 2000; Zou et al. 2000), has been demonstrated using these vectors (Schiedner et al. 1998). Retrovirus Retroviruses are RNA viruses that replicate through a DNA intermediate (Kay et al. 2001). They were among the first viruses to be developed for gene therapy and have subsequently become the most