**2. Gene therapy challenges and dilemmas**

The principle of gene therapy possesses undeniable therapeutic advantages over the conventional therapeutic modalities that are basically dependent upon exploitation of small molecules or biological pharmaceuticals. These advantages are: 1) specific or selective treatment of diseased cells/tissue, 2) minimal adverse consequences, 3) correction of the genetic cause of a disease, and 4) long-term treatment after single application (Rubanyi, 2001). Basically, to silence/suppress a target gene or to correct a genetic defect, the gene-based therapeutics such as oligodeoxynuleutides (ODNs), plasmid DNA, ribozymes, DNAzymes or short interfering RNA (siRNA) need to be shuttled to the target site. Delivery of gene-based therapeutics has been also advanced by development and implementation of various strategies, including: biological (e.g., viral vectors), physical (e.g., microinjection and electroporation, gene gun, ultrasound, and hydrodynamic delivery), and chemical (e.g., nonviral vectors) approaches. However, gene transfer into various target cells still faces major obstacles including poor delivery efficiency, cellular toxicity, immunogenicity and oncogenicity, as well as short-term transgenic expression and poor expression levels.

The first clinical test of gene therapy was accomplished a decade ago with the transfer of the missing "adenosine deaminase" gene into lymphocytes isolated from patients with severe combined immune-deficiency syndrome (i.e., as ex-vivo gene therapy approach). However, despite the early promising prophecy on the high effectiveness of gene therapy, the existing clinical experience indicate insufficient therapeutic efficacy coupled with increasing safety concerns and ethical issues (Verma & Somia, 1997). In some cases, aptamer-based genomedicines (e.g., Pegaptanib sodium, Macugen™) have been successfully utilized for treatment of the age related macular degeneration (Barar et al., 2008). Gendicine™ is an adenoviral p53-based gene medicine that was approved by the Chinese FDA in 2003 for treatment of head and neck cancer, while Advexin™ (a similar gene therapy approach from Introgen) was turned down by the US FDA in 2008. In fact, the death of Jesse Gelsinger in a gene therapy experiment in 1999 imposed a significant setback to gene therapy research in the United States, however many scientists aimed to resolve problems associated with the gene therapy strategies. In 2006, an international group of scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells (Ott et al., 2006). Also in 2007, the world's first gene therapy trial for inherited retinal disease was announced for treatment of Leber's congenital amaurosis which is a inherited blinding disease caused by mutations in the RPE65 gene (Maguire et al., 2008). It should be evoked that the performance and pathogenicity of viral vectors (e.g., retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses) and nonviral vectors have been evaluated in animal models. Promising results form the basis for clinical trials to treat genetic disorders and acquired diseases, however vector development/advancement remains a seminal concern for improved gene therapy technologies (Verma & Weitzman, 2005). Fundamentally, an ideal gene delivery method should protect the transgene against degradation, transport the transgene into the cytoplasm and then nucleus of target cells with little undesired detrimental effects (Gao et al., 2007).

Results obtained from *in vitro* studies have revealed that treatment of cells with antisense oligonucleotides (As-ODNs) for a period of only a few hours can bestow the desired effects

"toxicogenomics" of the nonviral vectors using global gene expression profiling techniques

The principle of gene therapy possesses undeniable therapeutic advantages over the conventional therapeutic modalities that are basically dependent upon exploitation of small molecules or biological pharmaceuticals. These advantages are: 1) specific or selective treatment of diseased cells/tissue, 2) minimal adverse consequences, 3) correction of the genetic cause of a disease, and 4) long-term treatment after single application (Rubanyi, 2001). Basically, to silence/suppress a target gene or to correct a genetic defect, the gene-based therapeutics such as oligodeoxynuleutides (ODNs), plasmid DNA, ribozymes, DNAzymes or short interfering RNA (siRNA) need to be shuttled to the target site. Delivery of gene-based therapeutics has been also advanced by development and implementation of various strategies, including: biological (e.g., viral vectors), physical (e.g., microinjection and electroporation, gene gun, ultrasound, and hydrodynamic delivery), and chemical (e.g., nonviral vectors) approaches. However, gene transfer into various target cells still faces major obstacles including poor delivery efficiency, cellular toxicity, immunogenicity and

oncogenicity, as well as short-term transgenic expression and poor expression levels.

The first clinical test of gene therapy was accomplished a decade ago with the transfer of the missing "adenosine deaminase" gene into lymphocytes isolated from patients with severe combined immune-deficiency syndrome (i.e., as ex-vivo gene therapy approach). However, despite the early promising prophecy on the high effectiveness of gene therapy, the existing clinical experience indicate insufficient therapeutic efficacy coupled with increasing safety concerns and ethical issues (Verma & Somia, 1997). In some cases, aptamer-based genomedicines (e.g., Pegaptanib sodium, Macugen™) have been successfully utilized for treatment of the age related macular degeneration (Barar et al., 2008). Gendicine™ is an adenoviral p53-based gene medicine that was approved by the Chinese FDA in 2003 for treatment of head and neck cancer, while Advexin™ (a similar gene therapy approach from Introgen) was turned down by the US FDA in 2008. In fact, the death of Jesse Gelsinger in a gene therapy experiment in 1999 imposed a significant setback to gene therapy research in the United States, however many scientists aimed to resolve problems associated with the gene therapy strategies. In 2006, an international group of scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells (Ott et al., 2006). Also in 2007, the world's first gene therapy trial for inherited retinal disease was announced for treatment of Leber's congenital amaurosis which is a inherited blinding disease caused by mutations in the RPE65 gene (Maguire et al., 2008). It should be evoked that the performance and pathogenicity of viral vectors (e.g., retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses) and nonviral vectors have been evaluated in animal models. Promising results form the basis for clinical trials to treat genetic disorders and acquired diseases, however vector development/advancement remains a seminal concern for improved gene therapy technologies (Verma & Weitzman, 2005). Fundamentally, an ideal gene delivery method should protect the transgene against degradation, transport the transgene into the cytoplasm and then nucleus of target cells with

Results obtained from *in vitro* studies have revealed that treatment of cells with antisense oligonucleotides (As-ODNs) for a period of only a few hours can bestow the desired effects

i.e. DNA microarray.

**2. Gene therapy challenges and dilemmas** 

little undesired detrimental effects (Gao et al., 2007).

of As-ODNs, while animal experiments demand repeated administration through multiple injections for prolonged exposure to As-ODNs. Despite promising results of some in vivo studies with free As-ODNs, improved delivery systems are essential to increase the efficacy of As-ODNs and to reduce its amount and frequency of administration (Hughes et al., 2001). Successful delivery of desired genes are important for both *ex vivo*, where cells undergo gene therapy in culture prior to implantation into the patient, or *in vivo* gene therapy where nucleic acids are administered directly to the patient to attain the desired gene change. Preferably, in either approach, only the therapy-intended gene expression changes should occur. However, this is not always the case, for example viral vectors are known to be efficient delivery systems for nucleic acids but can also induce immunogenic responses (Audouy et al., 2002; Ferber, 2001a; Ferber, 2001b). Hence, several nonviral gene delivery nanosystems such as cationic polymer- or lipid-based formulations have been developed for nucleic acid delivery. These cationic nanostructures can readily condense DNA into complexes and form polyplexes/lipoplexes to be used for *ex vivo* and *in vivo* gene therapy.

Although the CPs/CLs can principally enhance the delivery and improve the biological end-point of genomic-therapeutics, they often exert cytotoxicity depending on delivery system and target cell/tissue (Pedroso de Lima et al., 2001). Thus, both transfection efficiency evaluation and safety assessment are essential for gene transfer with these gene therapy vectors. A number of factors may affect the efficacy and safety of nonviral vectormediated gene transfer; in particular their structural properties and type of target cells and tissue. It should be noticed that as various target cells may display different responses, the transfection efficacy and safety of vectors should be carefully optimized upon types of target cells and target organs. Once transfection accomplished, specific attention should be given to the genotoxicity potentials of gene-based medicines. Surprisingly, no substantial information is available about the genomic signature of the cationic delivery systems. We have previously investigated the potential of the commercially available nonviral vectors (e.g., Polyamidoamine (PAMAM) dendrimers such as Polyfect™ (PF) and Superfect™ (SF)) and lipids (e.g., Lipofectin™ (LF) and Oligofectamine™ (OF)) on global gene expression within human epithelial A431 and A549 cells by exploiting the cDNA microarray technology (Barar et al., 2009; Omidi et al., 2003; Omidi et al., 2005a; Omidi et al., 2005b; Omidi et al., 2008). These investigations revealed occurrence of inadvertent nonspecific gene expression changes within target cells upon treatments with these cationic gene delivery nanosystems. These findings led us to screen series of lipid- or polymer-based non-viral vectors for their toxicogenomic and genomic toxicity potentials in target cells.

Fig. 1 represents schematic illustrations of polymer/lipid based micro/nano systems used for delivery of genes/drugs.
