**6. Methods for modification of large DNA molecules**

Gene therapy using genomic constructs entails engineering of large DNA fragments often of repetitive nature. For instance, marker genes and other useful sequences, able to confer extra-chromosomal maintenance, need to be added to vectors carrying large genomic fragments, without causing any rearrangements. In other cases, the entire genomic locus of a therapeutic gene may not be available in a single BAC vector and linking of the inserts of two or more BACs is necessary. The technical difficulty in performing such manipulations has hampered progress in this field for a long time. Only recently, efficient engineering methods have been developed allowing the consideration of BACs carrying genomic loci as gene therapy vectors.

#### **6.1 Addition of marker and small genes**

In most cases, the cloning of mammalian selectable markers and small reporter genes on the vector region of a BAC carrying a genomic insert by classic molecular biology procedures is limited by lack of convenient cloning sites on the vector, the possible presence of many restriction sites in the insert and the difficulty in manipulating large DNA molecules without affecting their integrity. Alternatives to classic molecular biology techniques using restriction enzymes are therefore required. For adding reporter genes and short sequences onto BACs, site specific homologous recombination mediated by the bacteriophage P1 derived Cre/*loxP* system is such an alternative.

The Cre protein recognizes and catalyses efficiently recombination only between specific *loxP* sites, which are present on all BAC vectors making a modification method based on them

Non-Viral Gene Therapy Vectors Carrying Genomic Constructs 15

BACs, allowing for the addition of the 70-kb alphoid array into any BAC and subsequent

formation of HACs in the appropriate cells (Kotzamanis et al., 2005).

Fig. 2. Addition of large arrays of alphoid DNA onto BACs by recombineering

There are many large human genes which are of the same order of size, or larger, than the average insert size of the BAC libraries and for these it is often difficult to find a single BAC spanning the entire gene with all its associated controlling elements. Gene therapy using the genomic loci of such genes would require the assembly of different sequences into a single BAC clone by linking together all available overlapping BAC clones spanning the desired region. Recombineering mediated by the Red system from the λ-prophage has been used to link two overlapping BACs (Kotzamanis & Huxley, 2004; Zhang & Huang, 2003) and linking has been shown to be precise without causing any rearrangements, including shifting of the reading frame of the therapeutic gene (Kotzamanis et al., 2009). As shown in Figure 3, the method comprises two rounds of homologous recombination to link the inserts of two overlapping BACs. In the first round, the inserts of the BACs are subcloned into modified BAC vectors (pBACLink vectors linearized by *Not*I) by homologous recombination at regions indicated as HomA, HomB and HomC (which are PCR amplified and cloned into the pBACLink vectors prior to their linearization). In the second round, one of the modified BACs is linearized by the rare cutting enzyme I-*Ppo*I and introduced into recombination efficient bacteria containing the other modified BAC, resulting in recombination at HomB and Cma (part of the chloramphenicol resistance gene present on all BACs) and linking of the two inserts in a single BAC. More overlapping BAC inserts can be added by alternating use of the two pBACLink vectors described in the study (Kotzamanis & Huxley, 2004).

**6.3 Linking of two overlapping BACs** 

generally applicable. As shown in Figure 1, the first step in such a method is the construction of a suitable retrofitting plasmid (pRetro) that carries the gene to be added onto the BAC, a *loxP* site and a selectable marker. Replication of pRetro depends on the high-copy gamma origin (γ-ori) that only operates in an *E. coli* host expressing the π protein (product of the *pir* gene). Following insertion of the plasmid into the BAC in the DH10B *E. coli* host which does not express π, the γ-ori becomes not functional and the BAC remains low-copy. The system shown in Figure 1 uses a separate plasmid to express Cre that is co-transfected with the retrofitting plasmid and then lost once retrofitting has occurred without being involved in the recombination process as it does not have a *loxP* site (Mejía & Larin, 2000). This plasmid contains a LacZ-cre fusion gene so that Cre is expressed after IPTG induction and also a temperature sensitive origin of replication that is functional only at 300C. As shown in Figure 1, *in vivo* retrofitting of the BAC with pRetro occurs at 300C and then bacteria are grown at 420C so that the Cre expressing plasmid is lost to avoid any further unwanted recombination events. Various pRetro plasmids for conveniently adding a G418 mammalian selectable marker, a GFP reporter gene, a luciferase reporter gene and/or an *OriP*/EBNA-1 element onto any BAC have been made available (Magin-Lachmann et al., 2003).

Fig. 1. Addition of small marked/reporter genes onto BACs by Cre/*loxP* recombination

#### **6.2 Addition of large sequences**

The construction of a genomic DNA-containing gene therapy vector with the ability to remain extra-chromosomally may involve the addition of large stretches of DNA that are difficult to clone into a pRetro plasmid in order to add them to a BAC by Cre/*loxP* recombination, as described in the previous section. For example, a 70-kb alphoid array has been shown to be required so as to enable a PAC vector to form HACs in HT1080 cells (Ebersole et al., 2000). For such applications, a method for manipulating large segments of DNA, based on homologous recombination in *E. coli* and termed recombineering, has been developed (Copeland et al., 2001). In recombineering, the sequence to be introduced is flanked by two regions of homology to the BAC, the length of which depends on the recombination system that catalyzes the recombination reaction. A selectable marker is also included in most applications so as to allow selection for correctly retrofitted clones in *E. coli*. The phage recombination systems RecET and Red consist of genes encoding proteins involved in homologous recombination of cryptic Rac prophage and bacteriophage λ respectively. These systems are relatively efficient, do not require long homology regions and rarely catalyze unwanted recombination events when used in recombineering (Court et al., 2002; Muyrers et al., 2001). Particularly the Red recombination system has been used to introduce a 70-kb alphoid array into a BAC, carrying a 156-kb genomic insert containing the *HPRT* gene, by recombineering (Figure 2) and expression of the *HPRT* gene from generated HACs has been demonstrated (Kotzamanis et al., 2005). As shown in Figure 2, recombination was targeted to the chloramphenicol resistance gene which is present on all

generally applicable. As shown in Figure 1, the first step in such a method is the construction of a suitable retrofitting plasmid (pRetro) that carries the gene to be added onto the BAC, a *loxP* site and a selectable marker. Replication of pRetro depends on the high-copy gamma origin (γ-ori) that only operates in an *E. coli* host expressing the π protein (product of the *pir* gene). Following insertion of the plasmid into the BAC in the DH10B *E. coli* host which does not express π, the γ-ori becomes not functional and the BAC remains low-copy. The system shown in Figure 1 uses a separate plasmid to express Cre that is co-transfected with the retrofitting plasmid and then lost once retrofitting has occurred without being involved in the recombination process as it does not have a *loxP* site (Mejía & Larin, 2000). This plasmid contains a LacZ-cre fusion gene so that Cre is expressed after IPTG induction and also a temperature sensitive origin of replication that is functional only at 300C. As shown in Figure 1, *in vivo* retrofitting of the BAC with pRetro occurs at 300C and then bacteria are grown at 420C so that the Cre expressing plasmid is lost to avoid any further unwanted recombination events. Various pRetro plasmids for conveniently adding a G418 mammalian selectable marker, a GFP reporter gene, a luciferase reporter gene and/or an *OriP*/EBNA-1 element onto

any BAC have been made available (Magin-Lachmann et al., 2003).

**6.2 Addition of large sequences** 

Fig. 1. Addition of small marked/reporter genes onto BACs by Cre/*loxP* recombination

The construction of a genomic DNA-containing gene therapy vector with the ability to remain extra-chromosomally may involve the addition of large stretches of DNA that are difficult to clone into a pRetro plasmid in order to add them to a BAC by Cre/*loxP* recombination, as described in the previous section. For example, a 70-kb alphoid array has been shown to be required so as to enable a PAC vector to form HACs in HT1080 cells (Ebersole et al., 2000). For such applications, a method for manipulating large segments of DNA, based on homologous recombination in *E. coli* and termed recombineering, has been developed (Copeland et al., 2001). In recombineering, the sequence to be introduced is flanked by two regions of homology to the BAC, the length of which depends on the recombination system that catalyzes the recombination reaction. A selectable marker is also included in most applications so as to allow selection for correctly retrofitted clones in *E. coli*. The phage recombination systems RecET and Red consist of genes encoding proteins involved in homologous recombination of cryptic Rac prophage and bacteriophage λ respectively. These systems are relatively efficient, do not require long homology regions and rarely catalyze unwanted recombination events when used in recombineering (Court et al., 2002; Muyrers et al., 2001). Particularly the Red recombination system has been used to introduce a 70-kb alphoid array into a BAC, carrying a 156-kb genomic insert containing the *HPRT* gene, by recombineering (Figure 2) and expression of the *HPRT* gene from generated HACs has been demonstrated (Kotzamanis et al., 2005). As shown in Figure 2, recombination was targeted to the chloramphenicol resistance gene which is present on all

BACs, allowing for the addition of the 70-kb alphoid array into any BAC and subsequent formation of HACs in the appropriate cells (Kotzamanis et al., 2005).

Fig. 2. Addition of large arrays of alphoid DNA onto BACs by recombineering
