**1. Introduction**

546 Non-Viral Gene Therapy

Wagner,U., du,B.A., Pfisterer,J., Huober,J., Loibl,S., Luck,H.J., Sehouli,J., Gropp,M.,

Wang,H. and Wettig,S.D. (2011) Synthesis and aggregation properties of dissymmetric

Wang,M. and Thanou,M. (2010) Targeting nanoparticles to cancer. *Pharmacol. Res.* 62, 90-99. Weiss,J.M., Subleski,J.J., Wigginton,J.M. and Wiltrout,R.H. (2007) Immunotherapy of cancer by IL-12-based cytokine combinations. *Expert. Opin. Biol. Ther.* 7, 1705-1721. Wettig,S.D., Badea,I., Donkuru,M., Verrall,R.E. and Foldvari,M. (2007a) Structural and

Wettig,S.D. and Verrall,R.E. (2001) Thermodynamic Studies of Aqueous m-s-m Gemini

Wettig,S.D., Wang,C., Verrall,R.E. and Foldvari,M. (2007b) Thermodynamic and aggregation

Whitmore,M., Li,S. and Huang,L. (1999) LPD lipopolyplex initiates a potent cytokine

Wilson,R.F. (2010) The death of Jesse Gelsinger: new evidence of the influence of money and

Wojtowicz-Praga,S. (1997) Reversal of tumor-induced immunosuppression: a new approach

Xu,F., Li,S., Li,X.L., Guo,Y., Zou,B.Y., Xu,R., Liao,H., Zhao,H.Y., Zhang,Y., Guan,Z.Z. and

Yarom,N. and Jonker,D.J. (2011) The role of the epidermal growth factor receptor in the mechanism and treatment of colorectal cancer. *Discov. Med.* 11, 95-105. Yong,K.T., Hu,R., Roy,I., Ding,H., Vathy,L.A., Bergey,E.J., Mizuma,M., Maitra,A. and

Young,L.S., Searle,P.F., Onion,D. and Mautner,V. (2006) Viral gene therapy strategies: from

Yu,M.K., Park,J., Jeong,Y.Y., Moon,W.K. and Jon,S. (2010) Integrin-targeting thermally cross-

Zhao,Y., Zhao,L., Zhou,L., Zhi,Y., Xu,J., Wei,Z., Zhang,K.X., Ouellette,B.F. and Shen,H.

Zhang,L. (2009) Phase I and biodistribution study of recombinant adenovirus vector-mediated herpes simplex virus thymidine kinase gene and ganciclovir administration in patients with head and neck cancer and other malignant tumors.

Prasad,P.N. (2009) Tumor targeting and imaging in live animals with functionalized semiconductor quantum rods. *ACS Appl. Mater. Interfaces.* 1, 710-719.

linked superparamagnetic iron oxide nanoparticles for combined cancer imaging

(2010) Quantum dot conjugates for targeted silencing of bcr/abl gene by RNA interference in human myelogenous leukemia K562 cells. *J. Nanosci. Nanotechnol.* 10,

(AGO-OVAR 2.6). *Gynecol. Oncol.* 105, 132-137.

Surfactant Systems. *J. Colloid Interface Sci.* 235, 310-316.

response and inhibits tumor growth. *Gene Ther.* 6, 1867-1875.

prestige in human research. *Am. J. Law Med.* 36, 295-325.

basic science to clinical application. *J. Pathol.* 208, 299-318.

and drug delivery. *Nanotechnology.* 21, 415102.

delivery. *Phys. Chem. Chem. Phys.* 9, 871-877.

to cancer therapy. *J. Immunother.* 20, 165-177.

*Cancer Gene Ther.* 16, 723-730.

5137-5143.

*Chem. Chem. Phys.* 13, 637-642.

*J. Gene Med.* 9, 649-658.

Stahle,A., Schmalfeldt,B., Meier,W. and Jackisch,C. (2007) Gefitinib in combination with tamoxifen in patients with ovarian cancer refractory or resistant to platinumtaxane based therapy--a phase II trial of the AGO Ovarian Cancer Study Group

phytanyl-gemini surfactants for use as improved DNA transfection vectors. *Phys.* 

transfection properties of amine-substituted gemini surfactant-based nanoparticles.

properties of aza- and imino-substituted gemini surfactants designed for gene

To date, both viral and nonviral vectors have been exploited for delivery of gene-based therapies to target cells/tissues. Despite high efficiency of the viral vectors (e.g., retroviruses and adenoviruses), these vectors appear to be immunogenic and potentially harmful when used in clinical gene therapy protocols (Ferber, 2001b). Besides, the preparation and purification of the viral vectors appear to be laborious, cost-prohibitive and not amenable to industrial-scale manufacture. Nonviral vectors such as cationic lipids (CLs) and cationic polymers (CPs) have been categorized as advanced materials and their low immunogenicity, lack of pathogenicity, and ease of pharmacologic production continue to make them attractive alternatives to viral vectors (Medina-Kauwe et al., 2005). However, these vectors may also suffer from relatively low levels of gene transfer compared to viruses. Thus, the drive to advance these vectors continues resulting in considerable progresses in improved transfection efficiency. Nonviral vectors (in particular cationic gene delivery systems) are able to bind and enter the target cells, however they yield low gene expression. No substantial information is available on interactions of these vectors with cellular biomolecules. Since these medicaments tend to act at genomic levels, thus understanding the genomic impacts of the nonviral vectors may help develop more efficient gene delivery systems. Nonetheless, this needs recruitment of high throughput screening methodologies.

To date, exploitation of the "omics" concepts (e.g., genomics, proteomics and metabolomics) is going to change the face of pharmacotherapy towards significantly more advanced and efficient pharmaceuticals (e.g., gene based nanomedicines) with minimal adverse consequences (Aardema & MacGregor, 2002). Enormous efforts have also been devoted for application of the global gene expression profiling in pharmacologic and toxicological investigations. The gene expression profiling technology has been primarily exploited for identification of underlying mechanisms for toxicity of pharmaceuticals and their genomic signatures, by which the safety liabilities can be determined and manifestations of undesired genotoxicity can be prohibited (Suter et al., 2004; Yang et al., 2004).

This methodology can be successfully used for the discovery and development of any chemicals and pharmaceuticals including gene delivery nanosystems. The main focus of the current book chapter is to provide some useful information about "genocompatibility" and

Toxicogenomics of Nonviral Cationic Gene Delivery Nanosystems 549

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

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

Fig. 1 represents schematic illustrations of polymer/lipid based micro/nano systems used

For achievement of an efficient systemic delivery of gene-based nanomedicines, various factors appear to play crucial role, including: 1) the physicochemical characteristics of the gene-based therapies, 2) the effects of biological environment, 3) the functionality of

membranes and barriers, and 4) the biological impacts of cellular microenvironment.

**3. Cellular trafficking and toxicity of polycationic nanostructures** 

therapy.

toxicity potentials in target cells.

for delivery of genes/drugs.

"toxicogenomics" of the nonviral vectors using global gene expression profiling techniques i.e. DNA microarray.
