**2.1 RNA interference**

RNA interference (RNAi), the Nobel Prize winning mechanism for gene silencing (Fire *et al.*, 1998), raises nowadays increasing attention of many researchers as a new way to treat lifethreatening diseases like cancer (Akhtar, 2006) or other genetic disorders like cystic fibrosis (Griesenbach and Alton, 2009) or viral infection as respiratory syncytial virus (RSV) (Ge *et al.*, 2004) and as an in vitro research tool to investigate mechanisms which are involved in those diseases. Small interfering RNA (siRNA) duplexes of 19-23 base pairs could trigger sequence specific gene silencing in mammalian cells (Caplen *et al.*, 2001; Elbashir *et al.*, 2001; Hannon and Rossi, 2004; Meister *et al.*, 2004; Mello and Conte, 2004). The siRNAs are double stranded molecules, consisting of a guide strand that is perfectly complementary to a target mRNA and a passenger strand. Core components of this siRNA-mediated posttranscriptional silencing include the RNAse III enzyme Dicer and its co-factor transactivating response RNA-binding protein (TRBP) along with the Argonaute family of proteins, in particular Argonaute 2 (Ago 2) (Meister *et al.*, 2004), which is the catalytic engine of the RNA induced silencing complex (RISC). Dicer converts dsRNA into 21-25 nucleotide duplexes with 3' 2nt overhangs. The siRNA is incorporated into one or more of the Argonaute proteins in RISC for sequence specific target degradation or translational inhibition (Tuschl *et al.*, 1999). In general, perfect or near perfect base pairing between the siRNA guide strand and the target mRNA is required for Ago2 cleavage to occur. In

Toxicity of Polymeric-Based Non-Viral Vector Systems for Pulmonary siRNA Application 483

Pancreatic

abbreviations used: AMD: age related macular degeneration; APOB: apolipoprotein B; CNV: choroidale neovascularization; DME: diabetic macular edema; i.v.: intravenous; KSP: kinesin

ribonucleotide reductase M2 polypeptide; RSV: respiratory syntical virus; VEGF: vascular

laboratory work and in clinical trials siRNAs are most often chemically synthesized, bypassing the Dicer cleavage step for entry into RISC and avoiding any immune responses and toxicity which is described for long double stranded RNAs (dsRNAs)

RNAi has widely been used in drug development and several phase I and II clinical trials (Table 1) are ongoing. However, for therapeutic applications still some concerns and challenges need to be overcome, e.g. off-target effects, innate immune response and most

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

cancer Phase I

miniature biodegradable polymeric matrix, placed in the tumor using an endoscopic ultrasound biopsy needle


Silenseed Ltd

endothelial growth factor

(Behlke, 2008).

siG12D LODER (Local Drug EluteR)

KRAS G12D

Table 1. Summary of ongoing clinical trials for siRNA delivery,

importantly specific delivery into the cytoplasm of target cells.

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

source: http://clinicaltrials.ifpma.org/no\_cache/en/search-trials-ongoing/all/index.htm,

spindle protein; PC: pachyonychia congenital; PKN3: protein kinase N3; RRM2:



source: http://clinicaltrials.ifpma.org/no\_cache/en/search-trials-ongoing/all/index.htm,

Table 1. Summary of ongoing clinical trials for siRNA delivery,

abbreviations used: AMD: age related macular degeneration; APOB: apolipoprotein B; CNV: choroidale neovascularization; DME: diabetic macular edema; i.v.: intravenous; KSP: kinesin spindle protein; PC: pachyonychia congenital; PKN3: protein kinase N3; RRM2: ribonucleotide reductase M2 polypeptide; RSV: respiratory syntical virus; VEGF: vascular endothelial growth factor

laboratory work and in clinical trials siRNAs are most often chemically synthesized, bypassing the Dicer cleavage step for entry into RISC and avoiding any immune responses and toxicity which is described for long double stranded RNAs (dsRNAs) (Behlke, 2008).

RNAi has widely been used in drug development and several phase I and II clinical trials (Table 1) are ongoing. However, for therapeutic applications still some concerns and challenges need to be overcome, e.g. off-target effects, innate immune response and most importantly specific delivery into the cytoplasm of target cells.
