**3. Small interfering RNAs (siRNA)**

482 Non-Viral Gene Therapy

RSV

Pachyonychia

Delayed graft function,

Kidney transplantation

Chronic optic nerve atrophy Non-Arteritic Anterior Ischemic Optic Neuropathy

> Hypercholesterolemia

Pediatric RSV Phase IIb

cancer Phase I

congenita Phase Ib

AMD Phase II

Phase I

Phase I

(Cand5) VEGF AMD, DME Phase II

RSV Pediatric RSV

CALAA-01 RRM2 Solid tumor

K6a

(Sirna-027) VEGFR1 AMD, CNV &

Disorder Status Administration

VEGF Liver cancer Phase I i.v., free siRNA -

solid cancer Phase I i.v., free siRNA -

/ Formulation Remarks







study has been terminated due to potential for immune stimulation to interfere with further dose escalation

intravitreal injection, free siRNA

aerosolized siRNA, free siRNA

i.v., Cyclodextrinadamantan-PEGtransferrin nanocomplex.

Injection into a callus on the bottom of one foot, free siRNA

intravitreal injection, free siRNA

Phase II i.v., free siRNA -

intravitreal injection, free siRNA

Phase I i.v., liposomal

formulation

Company siRNA Target Disease/

ALN-VSP02 KSP and

Therapeutics Atu027 PKN3 Advanced

TD101 PC keratin

AGN211745

I5NP

(QPI-1002) p53

QPI-1007 Caspase 2

PRO-040201 APOB

Bevasiranib

ALN-RSV-01 ALN-RSV-02

Acuity Pharmaceuticals (Opko Health)

Alnylam Pharmaceuticals

Silence

Sirna Therapeutics (Calando Pharmaceuticals)

Sirna Therapeutics (TransDerm Inc.)

> Sirna Therapeutics

Quarks Pharmaceuticals

Tekmira Pharmaceuticals Corporation

siRNAs are very attractive for therapy because they are easily designed and synthesized, and their versatility allows simultaneous use of multiple siRNAs or change of sequences to accommodate virus mutations. The negative charge of siRNA and their size of around 14 kDa make it difficult to cross the cell membrane without any carrier. There are various delivery strategies under investigation, which includes nanoparticular systems consisting of polymers and/or lipids of different compositions and with or without any conjugation like antibodies or ligands for achieving the most specific way to the target side of action. Davis et al. showed 2008 first evidence for RNAi mechanism of action in human with their selfassembling, cyclodextrin polymer-based nanoparticle system (CALAA-01) targeting the riboucleotide reductase subunit 2 (RRM2) which could be used for therapy of different types of cancers (Heidel *et al.*, 2007; Davis, 2009; Davis *et al.*, 2010). At the same time Zimmermann, MacLachlan and colleagues reported successful siRNA delivery using a different approach for delivery (Zimmermann *et al.*, 2006). They introduced so-called stable nucleic acid lipid particles (SNALP) generated by ethanol dilution technique and showed for the first time in non-human primate a successful targeting of ApoB in the liver (Soutschek *et al.*, 2004; Morrissey *et al.*, 2005; Zimmermann *et al.*, 2006). Ge and co-workers (Ge *et al.*, 2004) used PEI 25 kDa to complex and protect siRNA specific to influenza virus genes and they showed successful reduction of influenza virus infection in mice. Alton et al. gave first evidence for successful gene therapy by using a lipid-based system to delivery CFTR DNA in cystic fibrosis patients (Alton *et al.*, 1999). Thus, gene therapy approaches still need improvements

Toxicity of Polymeric-Based Non-Viral Vector Systems for Pulmonary siRNA Application 485

nanoparticles, known as lipoplexes, of 50-200 nm in diameter (Sitterberg *et al.,* 2010). Interaction with serum components represents one of the major hurdles that influence the performance when used systemically (Zuhorn *et al.*, 2007). Recently, lipid-mediated delivery of siRNA against apolipoprotein B (ApoB) has been used to target ApoB mRNA to the (Soutschek *et al.*, 2004; Zimmermann *et al.*, 2006). The in vivo use of cationic lipids especially by i.v. administration presents significant problems as these reagents can be quite toxic. Despite problems with i.v. use, cationic lipids are employed for i.p. injection (Verma *et al.*, 2003; Flynn *et al.*, 2004; Miyawaki-Shimizu *et al.*, 2006), for CNS injection (Hassani *et al.*, 2005; Luo *et al.*, 2005) or in topical epithelial surface application (Maeda *et al.*, 2005; Palliser *et al.*, 2006) and intratracheal (Griesenbach *et al.*, 2006). Toxicity varies with the precise chemical composition of the lipids employed dose, and the delivering route. Variations in chemical composition can have a large impact on the functional properties of cationic lipid mixtures (Spagnou et al., 2004), and lipoplex/liposomal preparations have been devised with decreased toxicity that are more compatible with i.v. administration. Liposomes can be modified with ligands such as folate or small peptides, which assist with delivery and help target specific cell types or tissues (Meyerhoff, 1999; Dubey *et al.*, 2004). Through the use of neutral polyethylene glycolsubstituted surfaces and other approaches, liposomes can be stabilized and made more "stealthy" showing reduced clearance and improved pharmacokinetics (Oupicky *et al.*, 2002; Moghimi and Szebeni, 2003). These kinds of lipid nanoparticles have been successfully used to deliver antisense oligonucleotides and siRNAs in vivo (Braasch *et al.*, 2003; Chien *et al.*, 2005). Similar to the lipid-based non viral vector systems, the positive charges of polycations allow an efficient interaction with siRNAs to form so-called polyplexes, which can bind onto cell plasma membrane and be endocytosed. In contrast to the lipid-based systems that rely on the fusogenic property of the liposomes to mediate endosomal escape, polymeric carriers such as poly(ethylene imine) (PEI) use the so-called "proton-sponge" effect to enhance endosomal release of endocytosed polyplexes (Boussif *et al.*, 1995; Behr, 1997; Akinc *et al.*, 2005; Demeneix and Behr, 2005; Nel *et al.*, 2009). According to this mechanism, the deprotonated amines with different pKa values confer a buffer effect over a wide range of pH. This buffering may protect the siRNA from degradation in the endosomal compartment during maturation of the early endosomes to late endosomes and their subsequent fusion with the lysosomes. The buffering property also allows the polycation to escape from the endosome. At lower pH the buffering capacity causes an influx of chloride ions and water into the endosomes, which burst due to osmotic pressure and facilitating intracellular release of PEI - siRNA polyplexes. PEI has been used for many years to facilitate nucleic acid delivery (Boussif *et al.*, 1995; Demeneix and Behr, 2005). However, due to toxicity and variable performance it has not found generalized acceptance as a delivery tool for either antisense oligonucleotides or siRNAs. Nevertheless, PEI can be used as a prototype for formulation of more complex particles

Polyethylene imine (PEI) is a simple repetition of the 43 Da CH2-CH2-NH ethylene imine motifs. It can be synthesized from ethylene imine (aziridine) via ring opening polymerization or by hydrolysis of poly(2-ethyl-2-oxazolium), leading to branched or linear polymeric backbones, respectively (Godbey *et al.*, 1999). PEI represents one of the most comprehensive investigated cationic polymer for gene delivery in vitro and in vivo (Godbey

with improved properties (Kim and Kim, 2009).

**5. PEI-based non-viral vector systems** 

regarding specific targeting and successful delivery of the nucleic acid but clinical trials are ongoing and preclinical testing are conducted for different kind of diseases (Table 1).

### **4. Non-viral vector systems for siRNA delivery**

RNA interference (RNAi) based therapeutics represent a fundamentally new way to treat human disease by addressing targets that are otherwise "undruggable" with existing medicines (Novina and Sharp, 2004; de Fougerolles *et al.*, 2007). The goal of RNAi-based therapy represents the activation of selective mRNA cleavage for efficient gene silencing. There are two possibilities to harness the endogenous pathway: either i) by using viral vector to express short hairpin RNA (shRNA) that resembles miRNA precursors, or (ii) by introducing siRNAs that mimic Dicer cleavage product into the cytoplasm. Synthetic siRNAs utilize the naturally occurring RNAi pathway in a manner that is consistent and predictable, thus making them particularly attractive as therapeutics. Since they enter RNAi pathway later, siRNAs are less likely to interfere with gene regulation by endogenous miRNAs (Jackson *et al.*, 2003; Grimm *et al.*, 2006). The most important characteristics for effective design and selection of siRNAs are potency, specificity, and nuclease stability. Two types of off-target effects need to be avoided or minimized: i) silencing of genes sharing partial homology to the siRNA and ii) immune stimulation induced by recognition of certain siRNAs by the innate immune system. The activation of the innate immune systems by siRNA could be induced by recognition of dsRNAs by the serine/threonine protein kinase receptor (PKR) (Schlee *et al.*, 2006). This pathway is normally triggered by dsRNAs that are more than 30 nucleotides long, but at higher concentrations also siRNAs may be able to activate this pathway resulting in global translational blockade and cell death. The potential to activate toll-like receptors (TLRs) in the endosomal compartment is more likely to occur after siRNA delivery due to recognition of specific nucleotide sequence motifs (e.g. GU) by TLRs. TLR activation could trigger the production of type I interferons and proinflammatory cytokines, and induce nuclear factor kappa B (NF-kB) activation (Hornung *et al.*, 2005; Judge *et al.*, 2005). For example, the presence of 2'-O-methyl modifications within the siRNA duplex could abrogate the binding to TLR7 in endosomes and abolish immunostimulatory response. In addition, these modifications also reduce sequencedependent off-target silencing and may be particularly beneficial in enhancing siRNA target specificity (Judge *et al.*, 2006; Robbins *et al.*, 2008; Robbins *et al.*, 2009).

Due to increasing mortality and morbidity caused by several lung diseases, RNAi strategies have attracted particular attention and the lung as target organ provides an attractive tool because of the accessibility via non-invasive routes, e.g. nasal or pulmonary applications. The clinical success of siRNA-mediated interventions critically depends upon the safety and efficacy of the delivery methods and agents. Naked siRNAs are degraded in human plasma with a half-life of minutes (Layzer *et al.*, 2004; Choung *et al.*, 2006). Thus, the search for optimized nanocarriers to deliver siRNA is still under intensive investigation. The negative charge and chemical degradability of siRNA under physiologically relevant conditions make its delivery a major challenge (Gary *et al.*, 2007). Depending on their origin, two types of positively charged carriers could be distinguished: i) lipid–based and ii) polymeric-based carrier systems. Both systems provided several advantages to deliver siRNA*.* Liposome formation agents like Lipofectamine 2000 (Dalby *et al.*, 2004; Santel *et al.*, 2006) and cardiolipin analogues (Chien *et al.*, 2005; Pal *et al.*, 2005) have been successfully used for the delivery of siRNA. Negatively charged nucleic acids and positively charged lipids spontaneously form

regarding specific targeting and successful delivery of the nucleic acid but clinical trials are

RNA interference (RNAi) based therapeutics represent a fundamentally new way to treat human disease by addressing targets that are otherwise "undruggable" with existing medicines (Novina and Sharp, 2004; de Fougerolles *et al.*, 2007). The goal of RNAi-based therapy represents the activation of selective mRNA cleavage for efficient gene silencing. There are two possibilities to harness the endogenous pathway: either i) by using viral vector to express short hairpin RNA (shRNA) that resembles miRNA precursors, or (ii) by introducing siRNAs that mimic Dicer cleavage product into the cytoplasm. Synthetic siRNAs utilize the naturally occurring RNAi pathway in a manner that is consistent and predictable, thus making them particularly attractive as therapeutics. Since they enter RNAi pathway later, siRNAs are less likely to interfere with gene regulation by endogenous miRNAs (Jackson *et al.*, 2003; Grimm *et al.*, 2006). The most important characteristics for effective design and selection of siRNAs are potency, specificity, and nuclease stability. Two types of off-target effects need to be avoided or minimized: i) silencing of genes sharing partial homology to the siRNA and ii) immune stimulation induced by recognition of certain siRNAs by the innate immune system. The activation of the innate immune systems by siRNA could be induced by recognition of dsRNAs by the serine/threonine protein kinase receptor (PKR) (Schlee *et al.*, 2006). This pathway is normally triggered by dsRNAs that are more than 30 nucleotides long, but at higher concentrations also siRNAs may be able to activate this pathway resulting in global translational blockade and cell death. The potential to activate toll-like receptors (TLRs) in the endosomal compartment is more likely to occur after siRNA delivery due to recognition of specific nucleotide sequence motifs (e.g. GU) by TLRs. TLR activation could trigger the production of type I interferons and proinflammatory cytokines, and induce nuclear factor kappa B (NF-kB) activation (Hornung *et al.*, 2005; Judge *et al.*, 2005). For example, the presence of 2'-O-methyl modifications within the siRNA duplex could abrogate the binding to TLR7 in endosomes and abolish immunostimulatory response. In addition, these modifications also reduce sequencedependent off-target silencing and may be particularly beneficial in enhancing siRNA target

ongoing and preclinical testing are conducted for different kind of diseases (Table 1).

**4. Non-viral vector systems for siRNA delivery** 

specificity (Judge *et al.*, 2006; Robbins *et al.*, 2008; Robbins *et al.*, 2009).

Due to increasing mortality and morbidity caused by several lung diseases, RNAi strategies have attracted particular attention and the lung as target organ provides an attractive tool because of the accessibility via non-invasive routes, e.g. nasal or pulmonary applications. The clinical success of siRNA-mediated interventions critically depends upon the safety and efficacy of the delivery methods and agents. Naked siRNAs are degraded in human plasma with a half-life of minutes (Layzer *et al.*, 2004; Choung *et al.*, 2006). Thus, the search for optimized nanocarriers to deliver siRNA is still under intensive investigation. The negative charge and chemical degradability of siRNA under physiologically relevant conditions make its delivery a major challenge (Gary *et al.*, 2007). Depending on their origin, two types of positively charged carriers could be distinguished: i) lipid–based and ii) polymeric-based carrier systems. Both systems provided several advantages to deliver siRNA*.* Liposome formation agents like Lipofectamine 2000 (Dalby *et al.*, 2004; Santel *et al.*, 2006) and cardiolipin analogues (Chien *et al.*, 2005; Pal *et al.*, 2005) have been successfully used for the delivery of siRNA. Negatively charged nucleic acids and positively charged lipids spontaneously form nanoparticles, known as lipoplexes, of 50-200 nm in diameter (Sitterberg *et al.,* 2010). Interaction with serum components represents one of the major hurdles that influence the performance when used systemically (Zuhorn *et al.*, 2007). Recently, lipid-mediated delivery of siRNA against apolipoprotein B (ApoB) has been used to target ApoB mRNA to the (Soutschek *et al.*, 2004; Zimmermann *et al.*, 2006). The in vivo use of cationic lipids especially by i.v. administration presents significant problems as these reagents can be quite toxic. Despite problems with i.v. use, cationic lipids are employed for i.p. injection (Verma *et al.*, 2003; Flynn *et al.*, 2004; Miyawaki-Shimizu *et al.*, 2006), for CNS injection (Hassani *et al.*, 2005; Luo *et al.*, 2005) or in topical epithelial surface application (Maeda *et al.*, 2005; Palliser *et al.*, 2006) and intratracheal (Griesenbach *et al.*, 2006). Toxicity varies with the precise chemical composition of the lipids employed dose, and the delivering route. Variations in chemical composition can have a large impact on the functional properties of cationic lipid mixtures (Spagnou et al., 2004), and lipoplex/liposomal preparations have been devised with decreased toxicity that are more compatible with i.v. administration. Liposomes can be modified with ligands such as folate or small peptides, which assist with delivery and help target specific cell types or tissues (Meyerhoff, 1999; Dubey *et al.*, 2004). Through the use of neutral polyethylene glycolsubstituted surfaces and other approaches, liposomes can be stabilized and made more "stealthy" showing reduced clearance and improved pharmacokinetics (Oupicky *et al.*, 2002; Moghimi and Szebeni, 2003). These kinds of lipid nanoparticles have been successfully used to deliver antisense oligonucleotides and siRNAs in vivo (Braasch *et al.*, 2003; Chien *et al.*, 2005). Similar to the lipid-based non viral vector systems, the positive charges of polycations allow an efficient interaction with siRNAs to form so-called polyplexes, which can bind onto cell plasma membrane and be endocytosed. In contrast to the lipid-based systems that rely on the fusogenic property of the liposomes to mediate endosomal escape, polymeric carriers such as poly(ethylene imine) (PEI) use the so-called "proton-sponge" effect to enhance endosomal release of endocytosed polyplexes (Boussif *et al.*, 1995; Behr, 1997; Akinc *et al.*, 2005; Demeneix and Behr, 2005; Nel *et al.*, 2009). According to this mechanism, the deprotonated amines with different pKa values confer a buffer effect over a wide range of pH. This buffering may protect the siRNA from degradation in the endosomal compartment during maturation of the early endosomes to late endosomes and their subsequent fusion with the lysosomes. The buffering property also allows the polycation to escape from the endosome. At lower pH the buffering capacity causes an influx of chloride ions and water into the endosomes, which burst due to osmotic pressure and facilitating intracellular release of PEI - siRNA polyplexes. PEI has been used for many years to facilitate nucleic acid delivery (Boussif *et al.*, 1995; Demeneix and Behr, 2005). However, due to toxicity and variable performance it has not found generalized acceptance as a delivery tool for either antisense oligonucleotides or siRNAs. Nevertheless, PEI can be used as a prototype for formulation of more complex particles
