In addition to retroviruses, adeno-associated virus (AAV) can also facilitate integration of the transgene into the host genome. Unlike retroviruses, AAV was found to integrate preferentially into a specific site on chromosome 19 (146). AAV is a naturally defective, nonpathogenic, single-strand human DNA parvovirus. For productive infection and viral replication, coinfection with helper viruses, e.g., adenovirus, herpesvirus, or vaccinia virus are required. In the absence of a helper virus, AAV establishes latency in the host by integrating itself into the host genome. AAV has a broad host range and is also able to infect both dividing and nondividing cells (147). Hence recombinant AAV (rAAV) vectors have been exploited as alternative vehicles for gene therapy.

AAV-based vectors (148) are simple to construct, requiring only that the viral inverted terminal repeat (ITR) (which are 145 nucleotides each) is upstream from the gene of interest. Other important viral genes like rep (involved in replication and integration) and cap (encoding structrual genes) can then be supplied in trans. One disadvantage with such rAAV vectors is that site-specific integration of the gene of interest into the host genome is not observed (96). This is probably because the rep gene, which is important for mediating site specific integration in the absence of helper viruses, is not included in the construct with the gene of interest. Nonetheless, rAAV has been successfully applied to the delivery of various genes into a variety of tissues and persistence of transgene expression in these nondividing tissues, was reported (149-154). Baudard et al. (96) demonstrated that in rapidly dividing cells, continuous selective pressure is necessary to sustain gene expression in cells. MDR1 was used as the selectable marker in this study. Being among the smallest DNA animal viruses (~20 nm in diameter), another disadvantage of the AAV system is its limited packaging capacity since it can accomodate only aproximately 4.7 kb of the gene of interest. As such, a small and efficient promoter would be required to drive the expression of large genes. One such promoter is the AAV p5 promoter, which, together with the ITR, forms a 263-base pair cassette capable of mediating efficient expression in a CF bronchial epithelial cell line (149,150). Baudard et al. further demonstrated that the reduction of the p5 promoter-ITR cassette to 234 bp was also able to promote efficient gene expression (96).

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