**10. Toxicogenomics of PEI-based non-viral vector systems**

Toxicogenomic and genotoxic information of non-viral vector systems is rare, but of great concern when nowadays focusing personalized medicine. Gene delivery systems should be

Ruiz *et al.*, 2009). Thus, for in vivo use a lot of effort should be made to avoid the high proinflammatory effects caused by the rupture or leakage of the endosome caused by PEI. Godbey classified PEI-mediated toxicity in an immediate toxicity, associated with free PEI and a delayed form, connected with cellular processing of PEI/DNA polyplexes (Godbey *et al.*, 2001). To form stable and protective PEI nucleic acid polyplexes an excess of PEI polymer is needed, 60-80% PEI remains in a free form after nucleic acid escape and is mainly attributed to PEI toxicity. The high positively charged PEI molecule is able to disrupt cell membranes, disruption of the endosome is on one hand favourable with respect to the intended cytoplasmatic delivery, but on the other hand disruption of other cell membranes (e.g., lysosomal membranes, mitochondrial membrane, plasma membrane) is not favourable as it will cause stress responses or even apoptotic or necrotic cell death. In this context it has been shown that PEI causes apoptosis in an unspecific manner in all kinds of cells (Beyerle *et al.*, 2010a; Merkel *et al.*, 2011) which should be avoided with regard to human use. Therefore, a purification approach of the PEI polymer before and after complexation with nucleic acid is one possibility to reduce PEI-related toxicity (Boeckle *et al.*, 2004; Werth, 2006; Fahrmeir *et* 

Espescially, when regarding the lung as target organ the activation of the inflammosome should be avoided. Lung targeting could in general be achieved by systemic delivery or pulmonary delivery. Pulmonary delivery enhances siRNA retention in the lungs, lowers the dose of siRNA required for efficient delivery, and therefore implicates reduced systemic toxic effects, and due to lower nuclease activity in the lung siRNA stability is increased. RNAi can be used to treat or prevent diseases affecting the lungs, such as lung cancer (Li and Huang, 2006; Tong, 2006; Jere *et al.*, 2008; Ren *et al.*, 2009; Zamora-Avila *et al.*, 2009), various types of respiratory infectious diseases (Ge *et al.*, 2004; Fulton *et al.*, 2009; DeVincenzo *et al.*, 2010), airway inflammatory diseases (Lee and Chiang, 2008; Seguin and

Beyerle and co-workers investigated the effects of PEGylation on cytotoxicity and cellcompatibility of different PEG-PEI copolymers in murine lung cell lines and found a clear

The higher the degree of PEGylation on PEI25kDa with low molecular weight PEG, the stronger was the reduction of cytotoxicity and oxidative stress, but the proinflammatory potential of PEI remained high (Beyerle *et al.*, 2010b). The same group evaluated the pulmonary toxicity of PEI/siRNA complexes and found at day three after intratracheal delivery still high numbers of neutrophils and high levels of proinflammatory cytokines in the airspace of polyplex treated mice (Beyerle *et al*., 2011a and Beyerle *et al*., 2011c). The higher inflammatory potential but lower toxicity of PEI modifications is still an issue to be overcome when targeting pulmonary diseases. There is an urgent need to balance the

Toxicogenomic and genotoxic information of non-viral vector systems is rare, but of great concern when nowadays focusing personalized medicine. Gene delivery systems should be

*al.*, 2007).

**9. Lung toxicity** 

Ferrari, 2009), and cystic fibrosis (Pison *et al.*, 2006).

efficacy and toxicity of such nucleic acid carriers.

**10. Toxicogenomics of PEI-based non-viral vector systems** 

structure-function relationship (Fig. 1).

Fig. 1. Structure-function-relationships of PEG-PEI copolymers Overview of the structure-function relationships of PEG modified PEI copolymers (B-C) in comparisonto PEI 25kDa (A) with regard to cytotoxic (v,w), oxidative stress (x,y) and proinflammatory responses (z). Arrows represent the up- or downregulation of the investigated molecules.

able to pass through biological membranes/barriers and transfer the desired information to target sites with minimal impact on the integrity of the target cell or tissue (Forrest and Pack, 2002; Omidi *et al.*, 2008). Viral vectors possess high efficacy accompanied by stimulation of the immune systems which is a limitation of these systems to deliver nucleic acids and human use. Therefore, non-viral vector systems should overcome these adverse side effects and represent safer and more efficient alternatives with improved bioavailability and reduced cellular toxicity in the clinics (Akhtar *et al.*, 2000; Somia and Verma, 2000; Panyam and Labhasetwar, 2003). It has been shown that cationic polymers and lipid-based transfection reagents could elicit cellular gene expression changes and complexation with siRNA increased these changes (Omidi *et al.*, 2003; Omidi *et al.*, 2005; Fedorov *et al.*, 2006; Hollins *et al.*, 2007; Tagami *et al.*, 2007; Tagami *et al.*, 2008). Beyerle et al. analyzed the expression changes of genes related to cytotoxicity, inflammation and oxidative stress in a pathway focused qRT-PCR array system upon treatment with different PEI-PEG copolymers in murine lung epithelial cells (LA-4 cell line) and could show that PEGylated PEI copolymers altered the gene expression profile on cost of upregulation of genes involved in inflammatory and oxidative stress processes while PEI 25 kDa mainly induced genes related to cytotoxicity and apoptosis (Beyerle *et al.*, 2010a). In addition, the potential of PEI and PEI-PEG copolymers to induce DNA damage and therefore their genotoxic potential was investigated in a lung epithelial cell line derived from the MutaMouse, but no indication for

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