**1. Introduction**

Gene therapy is the use of genes or DNA for the treatment of diseases. For the treatment of inherited disorders, DNA carrying a functional gene is introduced into the cells of a patient to reverse the defect of the corresponding malfunctioning endogenous gene. Previous genetic characterization of the disease and cloning of the gene that causes it are necessary. In most cases, the cDNA of the therapeutic gene is cloned into a bacterial plasmid under the control of a strong heterologous promoter (often of viral origin). However, such constructs, called mini-genes, lack introns, promoters, enhancers, and long-range controlling elements that precisely control the temporal and spatial expression of the endogenous gene.

For gene therapy of some diseases it is important to achieve expression of the therapeutic gene at specific levels. Expression at lower levels than normal might not be sufficient to correct the defect and at higher levels could result in undesirable effects. In other cases, tissue-specific expression may be very important. The elements responsible for controlled and tissue-specific expression of a gene usually lie within the introns and the sequences before and after the gene. Therefore, the use of genomic constructs which contain the introns and flanking DNA of the therapeutic gene is expected to be more effective than that of minigene constructs in gene therapy for certain genetic diseases where precise levels of the gene product are required (reviewed by (Pérez-Luz & Díaz-Nido, 2010)). Bacterial Artificial Chromosomes (BACs), originating from the human genome project, contain genomic loci of approximately 180 kb on average and cover the entire human genome (Osoegawa et al., 2001). These sequenced BACs can accommodate most genes along with their regulatory elements and can serve as tools in gene therapy using genomic constructs.

Gene therapy as a modern therapeutic tool should provide a permanent cure to the patient by long-term maintenance and expression of the administered gene. This can be achieved either by integration of the transgene into the natural chromosomes or by other mechanisms for its replication and nuclear retention.

One of the most important aspects of gene therapy is the choice of the vector that will deliver and express the corrective gene in the appropriate cells. Current vectors fall into two categories: viral and non-viral. Apart from determining the method of delivery, the type of vector also determines the fate of the therapeutic gene within the cells. For instance, the vector may have the ability to remain extra-chromosomally. Non-viral artificial chromosome vectors and adeno-associated viral, adenoviral, Herpes viral and EBV vectors are all

Non-Viral Gene Therapy Vectors Carrying Genomic Constructs 3

Inclusion of introns also allows correct function of genes that encode for different products through differential splicing, such as the immunoglobulin genes. Promoters that lie in the flanking DNA could be cloned and used in a cDNA construct. However, the introns and flanking DNA also contain other elements that can participate in the control of chromatin condensation and therefore influence gene expression. The most important of these

Enhancers increase transcription independently of orientation and distance. They can suppress silencing of transgenes that is usually observed when integration occurs in a condensed and therefore repressive heterochromatin region, a phenomenon called position effect (Martin & Whitelaw, 1996). Moreover, they can target transgenes to transcriptionally active nuclear compartments and prevent their localization near heterochromatin (Francastel, et al., 1999). LCRs are more complex and contain enhancers (Li et al., 1999). A general characteristic they have is the presence of DNAse I hypersensitive sites (DHS), where chromatin is not condensed and transcription factors can bind to their cognate

The importance of using genomic constructs rather than cDNA or mini-genes has been shown for several genes, both in cultured cells and in transgenic mice. Generally, the presence of promoters, enhancers, LCRs and other elements, located 5' or 3' of the gene or within introns, resulted in position-independent expression of the transgenes in the correct tissue, at proper levels and right times in contrast to the use of small transgenes carrying heterologous promoters. For example transgenic mice generated with long constructs that included all the known DHS 5' of the class II MHC *Ea* gene showed position-independent, copy number dependent expression of *Ea*. Shorter constructs lacking some of the DHS were subject to position effects (Carson & Wiles, 1993). Similar results were obtained when a 250 kb YAC carrying the genomic locus of the mouse tyrosinase gene was used to generate transgenic mice. The transgene was expressed at levels comparable to the endogenous gene, in the right tissues and proportional to copy number but independent of position (Schedl et al., 1993). Physiological expression of the human Huntington Disease gene has also been achieved from a YAC in transgenic mice containing a targeted disruption of the endogenous gene. Huntigtin is essential for development since its absence is lethal in mice but the human transgene was expressed in the correct tissue, at adequate levels and early enough in development to rescue the mice from embryonic lethality (Hodgson et al., 1996). Likewise, the human Friedrich ataxia (*FRDA*) gene expressed from a 188-kb BAC has been shown to rescue *FRDA* knockout mice from embryonic lethality (Sarsero et al., 2004). Transgenic mice were also generated with constructs covering the human β-globin locus, which is a model system for studying developmental regulation of gene clusters. The locus consists of five genes, ε, Gγ, Aγ, δ and β, the expression of which is tightly regulated both temporally and spatially. ε-globin is expressed at embryonic stages, Gγ- and Aγ- in the fetal liver and δ- and β-globin in the bone marrow of adults. An upstream LCR and a downstream DHS seem to control the expression of the genes. Transgenic mice generated by using a 160-kb BAC containing the entire human β-globin locus, exhibited proper developmental regulation and tissue specific expression of the globin genes (Huang et al., 2000). Interestingly, expression of a human *BCL2L10* (*Boo*) BAC transgene targeted at the *HPRT* locus in transgenic mice, followed the human pattern. Human *Boo* mRNA was detected in organs that had no murine *Boo* mRNA but were known to host *Boo* expression in humans, suggesting that human regulatory elements which were present in the BAC and absent in the mouse endogenous gene could drive tissue-specific expression in the mouse background (Heaney et al., 2004).

elements are simple enhancers and locus control regions (LCRs) (Lipps et al., 2003).

sequences allowing transgene expression.

examples of this type. In contrast, retroviral and lentiviral vectors integrate into the host genome (reviewed by (Verma & Weitzman, 2005)).

The majority of current gene therapy approaches are based on viral vectors due to their highly efficient delivery into cells. There are some examples of successful viral gene therapy clinical trials which had impressive clinical benefit for the patients (Cavazzana-Calvo et al., 2000). However, viral gene therapy has been subjected to criticism mostly because of two unfortunate events. In one case a patient with ornithine transcarbamylase deficiency treated with an adenoviral vector died due to provocation of an immune response (Raper et al., 2003). This death raised a safety issue that is hard to address, as human immune responses cannot be predicted pre-clinically. In another case, SCID-X1 patients treated with a retroviral vector developed a leukemia-like condition due to disruption of an endogenous oncogene by integration of the vector (Hacein-Bey-Abina et al., 2003a, 2003b). Since vector integration is random and uncontrollable, insertional mutagenesis is a general problem that all integrating viral vectors have.

Ideally, vectors suitable for gene therapy should meet four criteria. Firstly, they should be safe. In this context all vectors that arise from non-human sequences might cause adverse immune responses and are not suitable. Additionally, vectors that integrate into the host genome at random positions are also unsafe. Secondly, they could be efficiently delivered into cells. Viral vectors have an advantage in this respect but recently developed physical methods for non-viral DNA delivery (Reviewed by (Al-Dosari & Gao, 2009)) might prove to be equally effective. Thirdly, they should remain permanently in the cells and provide longterm expression of the transgene they carry. As random integration is excluded due to the first criterion, extra-chromosomally retained or site-specific integrating vectors might be an alternative solution. Fourthly, their cloning capacity should be high enough to allow them to carry fully functional genes with appropriate regulatory elements. Such intact genes, or gene clusters, can be very large and conventional molecular biology techniques will be inadequate for manipulating them. New technologies are therefore needed.

This chapter will focus on non-viral vectors containing entire genomic loci rather than minigenes. The necessity for using these constructs will become clear through several examples of preclinical work with integrating vectors conferring position independent expression from large transgenes. Key points on delivery of large naked DNA molecules into patients using physical methods will be covered. Emphasis will be given to *ex vivo* transfer of genomic constructs to cultured mammalian cells and nucleofection and bactofection as two promising methods for delivering large constructs will be analyzed. A review of all available vectors that allow extra-chromosomal maintenance of foreign DNA will be provided with an emphasis on the structure and potential application of EBV-based episomes, Human Artificial Chromosomes and Scaffod/Matrix Attachment Region (*S/MAR*) vectors as examples of non-integrating extra-chromosomally retained vectors. In addition, two systems for targeted integration at specific sites not associated with carcinogenesis will be described. The availability of powerful recombination-based methods for manipulating large vectors, a process called recombineering, will also be covered. Finally, an example on the development of genomic DNA containing vectors for gene therapy of Cystic Fibrosis using recombineering will be given.

#### **2. Advantages of use of genomic constructs in gene therapy**

The argument for using genomic constructs rather than cDNA in gene therapy is that they contain all the introns and flanking DNA which can confer full control of gene expression.

examples of this type. In contrast, retroviral and lentiviral vectors integrate into the host

The majority of current gene therapy approaches are based on viral vectors due to their highly efficient delivery into cells. There are some examples of successful viral gene therapy clinical trials which had impressive clinical benefit for the patients (Cavazzana-Calvo et al., 2000). However, viral gene therapy has been subjected to criticism mostly because of two unfortunate events. In one case a patient with ornithine transcarbamylase deficiency treated with an adenoviral vector died due to provocation of an immune response (Raper et al., 2003). This death raised a safety issue that is hard to address, as human immune responses cannot be predicted pre-clinically. In another case, SCID-X1 patients treated with a retroviral vector developed a leukemia-like condition due to disruption of an endogenous oncogene by integration of the vector (Hacein-Bey-Abina et al., 2003a, 2003b). Since vector integration is random and uncontrollable, insertional mutagenesis is a general problem that all

Ideally, vectors suitable for gene therapy should meet four criteria. Firstly, they should be safe. In this context all vectors that arise from non-human sequences might cause adverse immune responses and are not suitable. Additionally, vectors that integrate into the host genome at random positions are also unsafe. Secondly, they could be efficiently delivered into cells. Viral vectors have an advantage in this respect but recently developed physical methods for non-viral DNA delivery (Reviewed by (Al-Dosari & Gao, 2009)) might prove to be equally effective. Thirdly, they should remain permanently in the cells and provide longterm expression of the transgene they carry. As random integration is excluded due to the first criterion, extra-chromosomally retained or site-specific integrating vectors might be an alternative solution. Fourthly, their cloning capacity should be high enough to allow them to carry fully functional genes with appropriate regulatory elements. Such intact genes, or gene clusters, can be very large and conventional molecular biology techniques will be

This chapter will focus on non-viral vectors containing entire genomic loci rather than minigenes. The necessity for using these constructs will become clear through several examples of preclinical work with integrating vectors conferring position independent expression from large transgenes. Key points on delivery of large naked DNA molecules into patients using physical methods will be covered. Emphasis will be given to *ex vivo* transfer of genomic constructs to cultured mammalian cells and nucleofection and bactofection as two promising methods for delivering large constructs will be analyzed. A review of all available vectors that allow extra-chromosomal maintenance of foreign DNA will be provided with an emphasis on the structure and potential application of EBV-based episomes, Human Artificial Chromosomes and Scaffod/Matrix Attachment Region (*S/MAR*) vectors as examples of non-integrating extra-chromosomally retained vectors. In addition, two systems for targeted integration at specific sites not associated with carcinogenesis will be described. The availability of powerful recombination-based methods for manipulating large vectors, a process called recombineering, will also be covered. Finally, an example on the development of genomic DNA containing vectors for gene therapy of Cystic Fibrosis

The argument for using genomic constructs rather than cDNA in gene therapy is that they contain all the introns and flanking DNA which can confer full control of gene expression.

inadequate for manipulating them. New technologies are therefore needed.

**2. Advantages of use of genomic constructs in gene therapy** 

genome (reviewed by (Verma & Weitzman, 2005)).

integrating viral vectors have.

using recombineering will be given.

Inclusion of introns also allows correct function of genes that encode for different products through differential splicing, such as the immunoglobulin genes. Promoters that lie in the flanking DNA could be cloned and used in a cDNA construct. However, the introns and flanking DNA also contain other elements that can participate in the control of chromatin condensation and therefore influence gene expression. The most important of these elements are simple enhancers and locus control regions (LCRs) (Lipps et al., 2003).

Enhancers increase transcription independently of orientation and distance. They can suppress silencing of transgenes that is usually observed when integration occurs in a condensed and therefore repressive heterochromatin region, a phenomenon called position effect (Martin & Whitelaw, 1996). Moreover, they can target transgenes to transcriptionally active nuclear compartments and prevent their localization near heterochromatin (Francastel, et al., 1999). LCRs are more complex and contain enhancers (Li et al., 1999). A general characteristic they have is the presence of DNAse I hypersensitive sites (DHS), where chromatin is not condensed and transcription factors can bind to their cognate sequences allowing transgene expression.

The importance of using genomic constructs rather than cDNA or mini-genes has been shown for several genes, both in cultured cells and in transgenic mice. Generally, the presence of promoters, enhancers, LCRs and other elements, located 5' or 3' of the gene or within introns, resulted in position-independent expression of the transgenes in the correct tissue, at proper levels and right times in contrast to the use of small transgenes carrying heterologous promoters. For example transgenic mice generated with long constructs that included all the known DHS 5' of the class II MHC *Ea* gene showed position-independent, copy number dependent expression of *Ea*. Shorter constructs lacking some of the DHS were subject to position effects (Carson & Wiles, 1993). Similar results were obtained when a 250 kb YAC carrying the genomic locus of the mouse tyrosinase gene was used to generate transgenic mice. The transgene was expressed at levels comparable to the endogenous gene, in the right tissues and proportional to copy number but independent of position (Schedl et al., 1993). Physiological expression of the human Huntington Disease gene has also been achieved from a YAC in transgenic mice containing a targeted disruption of the endogenous gene. Huntigtin is essential for development since its absence is lethal in mice but the human transgene was expressed in the correct tissue, at adequate levels and early enough in development to rescue the mice from embryonic lethality (Hodgson et al., 1996). Likewise, the human Friedrich ataxia (*FRDA*) gene expressed from a 188-kb BAC has been shown to rescue *FRDA* knockout mice from embryonic lethality (Sarsero et al., 2004). Transgenic mice were also generated with constructs covering the human β-globin locus, which is a model system for studying developmental regulation of gene clusters. The locus consists of five genes, ε, Gγ, Aγ, δ and β, the expression of which is tightly regulated both temporally and spatially. ε-globin is expressed at embryonic stages, Gγ- and Aγ- in the fetal liver and δ- and β-globin in the bone marrow of adults. An upstream LCR and a downstream DHS seem to control the expression of the genes. Transgenic mice generated by using a 160-kb BAC containing the entire human β-globin locus, exhibited proper developmental regulation and tissue specific expression of the globin genes (Huang et al., 2000). Interestingly, expression of a human *BCL2L10* (*Boo*) BAC transgene targeted at the *HPRT* locus in transgenic mice, followed the human pattern. Human *Boo* mRNA was detected in organs that had no murine *Boo* mRNA but were known to host *Boo* expression in humans, suggesting that human regulatory elements which were present in the BAC and absent in the mouse endogenous gene could drive tissue-specific expression in the mouse background (Heaney et al., 2004).

Non-Viral Gene Therapy Vectors Carrying Genomic Constructs 5

Physical methods facilitate entry of naked DNA into the cells by creating temporary microdisruption of the cell membrane due to physical forces, such as hydrostatic pressure, electric pulse, ultrasound, laser irradiation, magnetic fields and particle bombardment. As of March 2011, naked plasmid DNA has been used in 18.7% (n=319) of clinical gene therapy

In early experiments, naked plasmid DNA (or RNA) was injected directly into different organs of different animals and expression of reporter genes was detected at the sites of injection. In mice, this method was used to deliver and express reporter genes in several organs including skeletal muscle (Wolff et al., 1990) and lung (Meyer et al., 1995). Subsequently, expression of therapeutic genes has been achieved using this method and some human clinical trials for limb ischemia (Morishita et al., 2010), erectile dysfunction (Melman et al., 2007), Duchenne/Becker muscular dystrophy (Romero et al., 2004)) have been based on naked DNA injection into tissues. Although these were phase I or IIa trials aiming mostly at assessing safety, some transgene expression and clinical improvement has

A non-invasive alternative to conventional needle injection is jet injection. This technology is based on a high velocity narrow jet of liquid containing the DNA, which is able to penetrate the skin and underlying tissues. It is powered by compressed air and penetration in a specific tissue can be controlled by adjusting the air pressure. So far, the major application of jet injection has been the development of DNA vaccines (Raviprakash & Porter, 2006). Thanks to technical improvements, the efficiency of delivery by jet injection has reached that of other non-viral methods and has been evaluated recently in a clinical trial on patients

Further progress in the field has led to the development of the so-called hydrodynamic injection, which is considered to be the most efficient non-viral gene delivery method in mice (Al-Dosari & Gao, 2009). According to the hydrodynamic method, a high volume saline solution of plasmid DNA is injected into the tail vein at high velocity. Initial studies have shown that this results in high levels of gene expression in the liver (Liu, et al., 1999; Zhang et al., 1999). The hydrodynamic injection into the tail vain has also been shown to work relatively well with large BAC DNA (Hibbitt et al., 2007; Magin-Lachmann et al., 2004). Moreover, local hydrodynamic delivery into rabbit liver using catheter-assisted perfusion (Eastman et al., 2002) and pressure-mediated delivery to rat kidney (Maruyama et al., 2002) and to limb muscle of mammals (Hagstrom et al., 2004) have been achieved. it remains to be seen whether similar hydrodynamic methods could be deployed in human

*In vivo* electroporation is the application of electrical pulses following local injection of DNA in the target tissue. This temporarily increases the cell membrane permeability and facilitates DNA uptake by a mechanism that remains unclear. Under optimal conditions the efficiency of plasmid DNA delivery by *in vivo* electroporation can approach that of viral methods but the efficiency decreases when larger DNA molecules are used. By using a variety of electrodes, ranging from needle to surface electrodes, electroporation has been shown to be effective at

**3.1 Physical methods for direct delivery to patients** 

been shown but the level of expression was low.

with melanoma and breast cancer (Walther et al., 2008).

**3.1.1 Injection** 

patients.

**3.1.2 Electroporation** 

trials (http://www.wiley.com/legacy/wileychi/genmed/clinical/).

Transgenic work has also been carried out with respect to cystic fibrosis (CF). The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (*CFTR*) which is normally expressed in specific tissues and shows precisely regulated expression thanks to some DHS that are found as far as 80 kb upstream of the first exon (Smith, et al., 1995). Previous experiments with small mini-gene constructs that obviously could not cover the whole genomic region showed some expression of *CFTR* in transgenic mice (Alton et al., 1993; Hyde et al., 1993) and low levels of transient correction in CF patients (Caplen et al., 1995). However, such constructs were not expressed sufficiently in the appropriate tissues to achieve clinical improvement in patients. A 320-kb YAC carrying the human *CFTR* gene rescued CFTR null mice (Manson et al., 1997). Gene expression followed the wild type mouse pattern except in some tissues such as the pyloric glands, Brunner's glands, epididymis and sublingual glands, presumably due to absence of a distant DHS in the YAC or lack of recognition of the human control elements by mouse transcription factors. More recently, correct temporal and spatial expression of the human *COL6A1* gene (Xing et al., 2007), the human Brain-Derived neurotropic factor (*BDNF*) gene (Koppel et al., 2009) and the porcine Growth Hormone gene (Tong et al., 2010) has been demonstrated in BAC transgenic mice.

The ability of genomic constructs to drive tissue- and time-specific expression, unlike cDNA and mini-genes, has made BAC transgenics an additional tool to knockout transgenics for the identification of potential regulatory elements within the locus of a gene. For instance, an enhancer within the locus of the tyrosinase-related family (*Tyrp1*) gene (Murisier et al., 2006) and a region responsible for tissue-specific expression within the locus of the *Neurogenin1* gene (Quiñones et al., 2010) have been discovered in transgenic mice generated with BACs.
