**4. Extra-chromosomal vectors**

Regardless of the type and size of the therapeutic gene (small cDNA versus large genomic DNA) and the delivery method (viral versus non-viral) to be used in a gene therapy protocol, efficient retention and long-term expression of the transgene is required so as to eliminate the need for re-administrations. Integration into the host genome has widely been used in gene therapy to fulfil this requirement. However, the dangers of integration due to insertional mutagenesis have become a widely publicised issue as a result of a clinical trial using a retroviral vector to treat X-linked severe combined immune deficiency (SCIDX1). In this trial some patients developed leukaemia due to deregulation of the growth-promoting LIM domain only 2 (*LMO2*) proto-oncogene caused by integration of the vector (Hacein-Bey-Abina et al., 2003a, 2003b). The safety concerns regarding uncontrolled integration of the therapeutic gene into the host genome have been strengthened by observations that there is a preference of integrating vectors for the regulatory regions of transcriptionally active genes (Bushman et al., 2005). Given the need for long-term expression and the problems associated with vector integration, vectors that persist in the nucleus by being maintained episomally without integrating could be highly advantageous. Three different systems have been employed to achieve extra-chromosomal maintenance of the vectors carrying the therapeutic gene: systems based on elements from the Epstein-Barr virus (EBV), artificial chromosomes and systems based on *scaffold/matrix attachment region* (*S/MAR*). All these systems have a high cloning capacity and can be used in combination with large genomic constructs.

Non-Viral Gene Therapy Vectors Carrying Genomic Constructs 11

satellite (alphoid) DNA and some marker genes into HT1080 cells. No telomeric sequences or an origin of replication have been shown to be required, probably due to generation of circular HACs and initiation of replication at origins found within the marker genes (Ebersole et al., 2000). HACs generated this way exist as single (or low copy) chromosomes in the nucleus and have a high mitotic stability (close to 100%) in the absence of selection. The potential use of these vectors in gene therapy has been demonstrated by expression of large therapeutic genes from them (Grimes et al., 2001; Mejía & Larin, 2000). Further advance was noted when efficient methods for manipulating large sequences of repetitive nature, such as alphoid DNA, were developed and used to generate HACs, as will be discussed later on (Kotzamanis et al., 2005). Nevertheless, several issues need to be solved before any clinical application. First, HACs have been shown to form efficiently only in HT1080 cells so far. Whether this is due to their inability to form in other cell lines has not been answered yet, but is limiting their use for *in vivo* gene therapy. Second, all HACs produced by *de novo* synthesis in HT1080 cells have been between 1 and 10 Mb in size, definitely smaller than native chromosomes, but larger than the input DNA, suggesting that unpredictable amplifications and rearrangements have occurred during their formation, which is not desired for safe gene therapy vectors. Third, other fates of the input DNA than formation of HACs have been observed and integration has not been excluded (Harrington et al., 1997). This would not be a problem in *ex vivo* therapy where individual clones expressing the gene of interest from a HAC could be isolated, but in *in vivo* gene therapy, any interaction of the input DNA with the endogenous chromosomes could have the same consequences as viral vectors have. Further research is necessary to increase the efficiency of

HAC formation so as to ensure that no integration events take place.

*S/MARs* are diverse sequences found in all eukaryotic genomes where they are involved in many aspects of chromatin function such as organization of chromatin into loops, which seems to be mediated by the interaction between *S/MARs* and the nuclear matrix (Heng et al., 2004). Experiments with a plasmid vector containing an *S/MAR* element isolated from

provide episomal maintenance of foreign DNA introduced into cells. This vector was able to replicate and remain episomally in CHO cells at low copy number for more than 100 generations in the absence of selection and with a mitotic stability of 98% (Piechaczek et al., 1999). It was later confirmed that the mitotic stability of the vector was provided through the interaction of the *S/MAR* with the nuclear matrix (Baiker et al., 2000). Interestingly, the *S/MAR* used seemed to prevent vectors from integrating into the host genome as integration events were observed in less than 1% of stably transfected clones (Jackson et al., 2006). The

demonstrated in several cell lines and in primary cells (Papapetrou et al., 2006) and also *in vivo* in genetically modified pigs (Manzini et al., 2006). Furthermore, the same *S/MAR* element has been introduced by site-specific homologous recombination to a BAC carrying 135 kb of the human low density lipoprotein receptor *(LDLR)* genomic locus and shown to provide low copy episomal maintenance in CHO *ldlr*-/- cells for more than 100 generations without selection and long-term expression of the transgene at high enough levels to

In summary, it seems that unlike viral episomal vectors which need to encode viral factors required for their function, S/MAR vectors achieve their replication and segregation by

*-* gene has revealed one more feature of *S/MARs*, their ability to

 *S/MAR* to provide replication and episomal retention has been

**4.3** *S/MAR* **vectors** 

the human interferon

ability of the interferon

β

β

completely restore LDLR function in these cells (Lufino et al., 2007).
