Cell Biol Toxicol (2010) 26:1–20 The first infectious clone of AAV serotype 2 (AA2) was established in 1982. It was used to generate the first recombinant AAV (rAAV) vectors (Hermonat and Muzyczka 1984) and has therefore been studied in great detail, and many mechanisms involved in AAV integration and latency have been elucidated (Gao et al. 2002). AAV2 is widely used to transduce neurons as it demonstrates neuron-specific expres- sion. However, more recently, multiple serotypes of wild-type AAV (12 human serotypes and more than 100 serotypes from nonhuman primates), each with a different targeting capacity, have now been identified (Daya and Berns 2008). Due to the diverse tissue tropisms and potential to evade preexisting antibodies against the common human AAV serotype 2, some of these AAV serotypes may have the potential to be developed as novel human gene therapy vectors (Coura and Nardi 2007). The use of the different AAV serotypes in a pseudotyping approach (the genome of one ITR serotype being packaged into a different serotype capsid) has allowed broad tissue tropisms, with greater gene expression being seen in muscle, retina, liver and heart (using AAV serotypes 1, 5, 8 and 9, respectively). Studies have also examined the transduction and expression character- istics of these serotypes in brain (Cearley et al. 2008), and AAV9 was shown to transduce a large number of cells with unique expression pattern. However, some tissues remain refractory to transduction using avail- able serotypes, presenting a major challenge for AAV- based gene therapy in clinically relevant tissues. Two approaches have been employed: (1) altering the direct targeting of rAAV vectors by either the insertion of small peptides or ligands directly into the viral capsid sequence (e.g. aiding targeting of endothelial cells; Stachler and Bartlett 2006; White et al. 2004) or by site-directed, insertional mutagenesis of the AAV2 capsid (Muzyczka and Warrington 2005; Shi et al. 2001) and (2) using an associating molecule, such as a bispecific antibody (Bartlett et al. 1999) or biotin (Arnold et al. 2006) which interacts with both the viral surface and specific cell surface receptor and mediated indirect targeting. The latter approach enables the coupling of different adaptors to the capsid, ablating the native targeting and enhancing specific targeting without significantly changing the capsid structure. Regardless of serotype, the production of rAAV vectors involves splitting the AAV genome between 3 two plasmids: (1) a recombinant AAV vector plasmid where the AAV capsid genes are replaced with the gene of interest, bracketed by ITRs and (2) a non- rescuable AAV helper plasmid encoding AAV capsid proteins. The production of recombinant AAV vectors also requires a helper virus (adenovirus or HSV) which allows viral replication, and density gradient centrifugation is typically used to separate recombi- nant AAV vectors from the adenoviral or HSV helper. The inclusion of ITRs in rAAV vector systems prevents integration into chromosome 19, and current rAAV vectors persist primarily as extrachromosomal elements (Afione et al. 1996; Schnepp et al. 2005). In addition, the small size of these vectors limits the insertion of gene expression cassettes, and gene expression is generally of slow onset due to the required conversion of the single-stranded AAV DNA into double-stranded DNA. Such limitations have been overcome by the generation of minimal expres- sion cassettes and self-complementary AAV2 vectors, respectively. The latter enable the rate-limiting second-strand DNA synthesis to be bypassed, en- hancing transduction of some organs and tissues such as liver (McCarty et al. 2001), muscle (Wang et al. 2003), brain (Fu et al. 2003) and retina (Yang et al. 2002). As a result, rAAV-based gene transfer vectors still represent one of the most promising gene therapy systems and gain increasing popularity. rAAV2 vectors have been tested in preclinical studies for a variety of diseases such as haemophilia, alpha1 anti- trypsin deficiency, cystic fibrosis, Duchenne muscular dystrophy, rheumatoid arthritis, age-related macular degeneration (Buch et al. 2008) and others. The studies so far have shown that AAV-based vectors, particularly the AAV2 serotype, are safe and efficient tools for gene transfer (further information on AAV vectors was given by Professor Michael Linden during this meeting and is also published in this special edition of Journal of Cell biology and Toxicology). Adenoviral vectors Adenoviruses (Ads) have been isolated from a large number of species and tissue types, and in humans, they cause mild respiratory illnesses and gastroenter- itis. Wild-type adenoviruses have a double-stranded, linear DNA genome of approximately 36 kb in length. Extensive splicing of the genome results in the