**5.2 Adeno-associated virus based vectors**

12 Non-Viral Gene Therapy

recruiting and interacting with host cell proteins. Moreover, unlike HAC vectors, *S/MAR* vectors do not need to undergo amplifications and rearrangements to function and are therefore of defined structure and composition. These unique safety properties of *S/MARs*

An alternative to the use of episomal vectors described above, that still satisfies both requirements for permanent transgene expression and elimination of genotoxic effects is the controlled integration of the therapeutic construct at a specific site in the host genome where no active genes are present. Several vector systems have been developed to achieve this, with each one of them having its own limitations (Voigt et al., 2008). From this variety of available vectors, only two types will be described based on their preference for specific sequences that already exist in the human genome, their potential for *in vivo* use and their

The *Streptomyces* phage ΦC31 integrase is an enzyme that can catalyze site-specific recombination between a phage attachment site (*attP*) and a bacterial attachment site (*attB*) resulting in integration of ΦC31 into the bacterial genome and initiation of the lysogenic phase of its life cycle (Groth & Calos, 2004). This integrase has been shown to be able to irreversibly integrate a single copy of foreign DNA, containing the *attP* site, into the human genome at native pseudo *att* sites found in the intergenic regions on human chromosomes

A series of studies have validated the potential of ΦC31 integrase-based vector systems in *ex vivo* gene therapy by demonstrating expression of different therapeutic genes in cultured cells including human embryonic stem cells (Thyagarajan et al., 2008) and in *in vivo* gene therapy by showing expression of different therapeutic genes in animal models, like the dystrophin gene in dystrophic mouse muscle (Bertoni et al., 2006). Evidence that the system can be used in gene therapy with genomic constructs has been provided by transgenic work in *Drosophila*, where the ΦC31 integrase has been used to integrate large DNA fragments of up to 133 kb into the genome (Venken et al., 2006). However, other studies have questioned the safety of such vectors by showing that stably expressed ΦC31 integrase could cause numerous chromosomal abnormalities in human cells (Liu et al., 2006) and that in some cases ΦC31-mediated integration is associated with chromosome rearrangements, probably due to recombination between cryptic *att* sites (Ehrhardt et al., 2006). Recently developed mutational derivatives of ΦC31 integrase that have higher integration efficiency and specificity, may eliminate the safety concerns for its use in gene

A very similar approach for site-specific integration is based on the utilization of transposase enzymes, with the *Sleeping beauty* and the *piggyBac* being the most thoroughly studied, which allow for the integration of foreign genes into genomic regions containing transposable elements. (Ivics & Izsvak, 2010). However, such systems are unlikely to prove useful for integration of large genomic constructs as their ability to transpose is significantly decreased when the insert length is increased, a phenomenon called "length-dependence"

make them very attractive for use in gene therapy with large genomic constructs.

**5. Vectors integrating at specific sites** 

**5.1 Phage integrase based vectors** 

therapy (Keravala et al., 2009).

(Atkinson & Chalmers, 2010).

ability to support integration of large genomic constructs.

and far from known oncogenes (Chalberg et al., 2005).

Some features of the Adeno-associated virus (AAV) can be exploited for site-specific integration of foreign DNA. AAV is a non-pathogenic virus with a 4.7 kb single-stranded DNA genome comprising two genes, *rep* and *cap* flanked by 145 bp palindromic sequences termed inverted terminal repeats (ITRs) (Srivastava et al., 1983). In the presence of a helper virus such as adenovirus or herpes simplex virus, AAV can undergo replication and enter its lytic cycle while in their absence AAV integrates into the human genome and becomes latent. The ITRs contain the sequences required for replication, packaging and integration of the virus and the *rep* gene encodes four regulatory proteins required for catalysis of integration into the human genome during latency. Integration occurs into a specific site on chromosome 19 called the AAVS1 (Kotin et al., 1992).

It has been shown that human transgenes flanked by ITRs can integrate into the AAVS1, with the minimal requirement for expression of viral Rep in cultured human cells (Cortés et al., 2008), and *in vivo* in transgenic mice carrying the human AAVS1 (Liu et al., 2010; Recchia et al., 2004) . The AAV system has also been shown to be able to integrate large genes of 100kb in size into the AAVS1 (Oehmig et al., 2007). These studies have demonstrated the effectiveness of AAV-based vectors but again safety issues have emerged. Integration of small transgenes has been detected in 10-30% of infected human cells in culture with only about half of the integrations occurring specifically at the AAVS1 (Recchia & Mavilio, 2006), suggesting that there would be a 50% probability of insertional mutagenesis in a gene therapy application. In addition, persistent expression of the viral Rep protein is toxic and can cause chromosome instability and mobilisation of the transgene (McCarty et al., 2004).
