Manipulating cells by RNA

Various groups have shown that synthetic siRNAs can be recognised and taken up into the cellular RNAi pathway in vitro and mediate RNAi not only in Drosophila cells but also in a variety of other species including human (Elbashir et al., 2001a, Elbashir et al., 2001c, Fire et al., 1998, Hammond et al., 2000, Nykanen et al., 2001). Elbashir et al. demonstrated that synthetic 21- and 22bp dsRNAs with 3’ overhangs mediate sequence- specific mRNA degradation in a Drosophila in vitro model (Elbashir et al., 2001c).

It has also been shown, that short hairpin RNAs (shRNAs) with a stem of 25 to 30 nucleotides, which are also recognised by the Dicer enzyme, silence target mRNA more efficiently that 21mer siRNAs (Kim et al., 2005b, Siolas et al., 2005). There are eight characteristics, which have been identified with siRNA functionality. These are mostly empirical guidelines, e.g. it was suggested that two 21 nucleotide sense and antisense oligonucleotides, which both harbour a 2-nt overhang on the 3’ terminus, should be designed to target a gene of interest. In order to save the siRNA from degradation through endonucleases the overhanging bp should be dT (deoxythymidines).

Furthermore, the siRNA sequence should not target any other mRNA sequence in the cells of interest (perform BLAST search on EST libraries) and should not be directed at introns, untranslated regions (UTRs), sequences less than 75bp away from the start codon and regions with a high G+C content (over 50%). Low internal stability at the 3’ end of the sense strand as well as a lack of inverted repeats are also important. In addition, sense

strand preferences at positions 3, 10, 13 and 19 should be observed, e.g. A at position 19 (Mittal, 2004, Reynolds et al., 2004). There are several online tools available for the selection of siRNA sequences (Pei and Tuschl, 2006).

RNAi is not only discussed as a powerful tool for functional genomics but also as a possibility for future therapeutic gene-silencing drugs (Shuey et al., 2002). One problem for the use of RNAi as a therapeutic method might be the activation of dsRNA-dependent protein kinase (PKR) by the introduction of short double-stranded RNA as this activation will lead to apoptosis in the target cells.

Dimerisation of PKR due to dsRNA binding leads to autophosphorylation and activation of PKR. The activated PKR can then phosphorylate substrates such as the eukaryotic translation initiation factor eIF2". Once the small subunit of eIF2" is phosphorylated, a signalling cascade is altered which leads to apoptosis (Gil and Esteban, 2000).

For in vivo therapy, safe delivery of siRNAs or shRNAs is a challenging obstacle. siRNAs could be transported into cells using cholesterol-conjugation, antibody-fusion, liposomes or viral expression vectors while microinjection is only relevant for in vitro assays (Dykxhoorn and Lieberman, 2006). The delivery of shRNA-expressing plasmids, targeting the liver, was successfully shown in mice by hydrodynamic tail-vein injection. If transient expression in non-dividing cells is favourable, nonintegrating vectors, like adenoviruses or herpesviruses, should be chosen. However, if dividing cells are targeted and stable, long-term expression is desired, retroviruses are the appropriate choice (Snove and Rossi, 2006). Paul et al. showed that RNAi can be induced in HeLa cells by introduction of synthetic duplex RNAs of around 20bp using liposome transfection. The RNAs were expressed using a human U6 small nuclear RNA promoter (Paul et al., 2002). Methods for promoter-based expression have already been successfully used for stable gene silencing in vitro. However in vivo application is more challenging as the silencing of specific genes might also induce unwanted side effects. Toxicity can be associated with delivery, shRNA expression or the sequence itself (Snove and Rossi, 2006). Paddison and colleagues described gene silencing in various mammalian cell lines after transfection with a plasmid containing an shRNA expression cassette under the control of a U6 promoter (Paddison et al., 2002).

The choice of promoters depends on the desired expression level. While pol II promoters are suitable for moderate expression levels, higher expression levels can be achieved by combining a pol III (U6) and a pol II (U1) promoter as the transcribed products have different export pathways. There is also the possibility of using an inducible or tissue-

specific expression system, e.g. transcriptional elements which are responsive to tetracycline or the Cre-lox system for permanent genetic changes (Snove and Rossi, 2006).

Although it was originally suggested that in human cells silencing is associated with DNA methylation (Morris et al., 2004), Ting et al. showed that effective transcriptional gene silencing in breast and colon cancer cells was independent of DNA methylation. Furthermore, it was also achieved in a cell line that was genetically modified and lacked the capacity to methylate DNA (Ting et al., 2005).

Furthermore, gene silencing by lentivirus-delivered shRNAs under a U6 promoter was successfully shown in HeLa as well as primary dendritic cells (Stewart et al., 2003). RNAi has also been used extensively in stem cell research over the last years. It was shown that the stem cell specific transcription factors Oct4 and Nanog play a significant role in stem cell self-renewal by using siRNAs or shRNAs, respectively, which were produced from lentiviral vectors to downregulate the gene expression. The promoters used were U6 and H1 respectively (Ivanova et al., 2006, Zaehres et al., 2005). Even though RNAi is a very attractive technique for functional genomics and human therapeutics, it was previously reported that oversaturation of endogenous small RNA pathways by high shRNA expression from a viral vector, lead to increased morbidity in mice (Barik, 2006, Grimm et al., 2006).

To summarise, RNA interference can be used in a variety of scientific backgrounds and future research has to be focused on applying RNAi in gene or cancer therapy.