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1.4.1. Posttranscriptional gene silencing

Posttranscriptional gene silencing (PTGS) defines a mechanism that serves to protect the genome from foreign DNA or RNA by sequence specific destruction of the nucleic acids. PTGS has been shown to play a critical role in normal cellular functions 111 _{and it appears to be a natural defence mechanism against viruses and}

transposable elements 112,113_{. It was first discovered in plants into which a transgene}

that was supposed to enhance the expression of the product encoded by the transgene was introduced, but instead led to the suppression of itself and of a homologous endogenous gene 114. Thus, this process can be induced by the introduction of exogenous nucleic acids which target cellular mRNA homologous to its sequence for cleavage, thereby silencing endogenous genes specifically 115. Antisense oligonucleotides (AS-ODN) and short interfering RNAs (siRNA) are examples of exogenous nucleic acids that switch on PTGS. However, there are other molecules that trigger specific mRNA cleavage such as ribozymes and which are also used for experimental purposes 115,116.

Ribozymes, like the hammerhead ribozyme, are RNA enzymes that can be used for gene knock-down. They consist of a catalytic domain flanked by sequences which are complementary to the target mRNA enabling selective binding. The target mRNA is cleaved directly by the intrinsic activity of the ribozyme (Fig. 1.8). However, the RNA backbone makes these molecules susceptible for degradation from RNases and thus biological unstable in vivo. Deoxyribozymes, which are catalytic DNAs, can also be applied for selective gene silencing. They bind to their target RNA and directly cleave it in the same way as the ribozymes do. In contrast to the ribozymes, no natural occurring deoxyribozyme is known to exist and they exhibit a greater biological stability due to their DNA backbone 117,118_.

Antisense oligonucleotides (AS-ODN) consist of approximately 15-20 desoxynucleotides and bind to the pre-mRNAs or the mRNAs homologous to its sequence 116. The AS-ODN can then either occupy critical sites of the mRNA thereby disturbing translation or target the mRNA for degradation 113,116. By constructing AS-ODNs that are targeted to the 5´terminus of the mRNA, the ribosome is blocked by steric hindrance and translation can not be completed 116,117. Most widely used is the mechansim of RNase H cleavage, which is induced by AS-ODNs that are negatively charged (Fig. 1.8). RNase H hydrolyses RNA strands when bound to a DNA strand and therefore causes the degradation of mRNA 116,117. Since naked DNA is rapidly degraded by exo- and endonucleases in the cell, the AS-ODN can be modified in different ways to increase its stability. The most common protection is the replacement of one of the non bridging oxygen atoms in the phosphate group with a sulfur group, giving a so called phosphorothioate oligonucleotide 115,116,118. This modification gives the AS-ODN a half-life in human serum of about 9-10 hours compared to approximately 1 hour for unmodified AS-ODN. In addition, these molecules are negatively charged and therefore water soluble and activate RNase H. A disadvantage with these molecules is that they exhibit a slightly reduced affinity towards complementary RNAs, but instead a greater specificity of hybridization compared to unmodified AS-ODN. Another disadvantage of phosphorothioate AS-ODN is the ability to bind to certain proteins, such as heparin-binding proteins. However, this shortcoming was proven to be of advantage in in vivo situations, where AS-ODNs bound plasma proteins which protected them from filtration and increased their serum half-life 116.

The use of short interfering RNAs (siRNA) to silence protein expression is expanding rapidly. This technique takes advantage of the process of RNA interference (RNAi), which was first discovered in Caenorhabditis elegans, Drosphila and plants 119 but has also been observed in different vertebrate systems 120 such as in early mice embryos, zebrafish and Xenopus 119. Naturally occurring RNAi is triggered by dsRNA that targets single-stranded RNA homologous in sequence to the dsRNA for degradation 119,120.

It has been shown that dsRNA is a potent inducer of nonspecific gene silencing in mammalian cells due to activation of the interferon response. These nonspecific responses have for quite a time disturbed the detection of RNAi in somatic mammalian cells 119,120. A breakthrough came when the nonspecific gene silencing of dsRNA in mammalian cells was shown to be length-dependent 119 and that this can be overcome by introduction of short interfering RNAs with less than 30 nucleotides, that can be either expressed endogenously (short hairpin RNA) or delivered exogenously 117,120. Endogenously expressed hairpin RNA (shRNA) is cut into shorter RNAs, by the cytosolic RNAse III enzyme Dicer 120. The short interfering RNAs (siRNA) assemble with the RNA-induced silencing complex (RISC) and target mRNA homologuous in sequence for degradation by RISC 120. Due to its helicase activity, RISC separates the two mRNA strands, thereby enabling the siRNA to bind to the target mRNA. RISC also exhibits an endonuclease activity which then hydrolyses the mRNA at the site of siRNA binding 121 (Fig. 1.8). Although reported to be the most potent antisense technology discovered so far, a major disadvantage with this technique is the off-target effects, i.e the non specific silencing of other proteins 122_.

This can partly be overcome by lowering the siRNA concentration, however at the expense of efficiency.

ds RNA Chemical syntethis Vector-based siRNA A) RISC Protein translation AS-ODN RNase H B) Ribozyme C)

Fig. 1.8 Strategies to suppress protein translation

Short interfering RNA (siRNA) can be produced by either cleavage by Dicer from longer dsRNA (A, top left), chemical synthesis (A, top middle) or transcription from a vector containing the target sequence (A, top right). In all cases, siRNA assembles with RISC and directs mRNA cleavage. AS-ODN binds to mRNA homologous in sequence and can either prevent translation by blocking the ribosome or induce mRNA cleavage by a RNase H dependent mechanism (B, bottom right). Ribozymes and Deoxyribozymes also bind mRNA homologous in sequence but exhibit an intrinsic catalytic activity and so silence the gene product (C, bottom left).

1.4.2. Magnetofection- a highly efficient tool for transfection of

endothelial cells

The challenge for all antisense based technologies is the efficient delivery of these molecules into cells, tissues or organs as the negative charge of RNA and DNA molecules limits the cellular uptake. A new method of introducing gene vectors into cells is the so called magnetofection method, invented by Plank and colleagues at the Institute of Experimental Oncology at the Technical University of Munich, Germany, where magnetic nanoparticles associated with vector DNA are transfected

into cells by the influence of an external magnetic field 123 (Fig. 1.9). This technique has been shown to enhance the efficiency of both viral or non viral vectors up to several thousand times 124.

Magnet Culture plate

AS-ODN coupled to magnetic beads

Fig. 1.9 The method of magnetofection

Magnetic nanoparticles coated with Polyethylenimine bind to DNA due to their positive charge. This complex is applied to and transfected into the cells by a short time (15 minutes) influence of an external magnetic field.

There are several advantages of using this transfection method. When performing transfection in vitro this method provides a quick and easy way to introduce DNA into cells. The incubation time of the DNA-nanoparticle complexes with the cells is greatly reduced (from standard 2-4 hours to 15 minutes) with a vast increase in vector concentration at the cell surface as the consequence. This in turn means that the vector dose needed for yielding a satisfying transfection level also can be reduced 124-126. Further, since these magnetic particles do not rely on receptors or other cell membrane bound proteins for cell uptake, it is possible to transfect cells that normally are non permissive 124. Importantly, the magnetofection method in combination with a standard lipid transfection reagent has been shown to successfully deliver AS-ODN to primary human umbilical vein endothelial cells (HUVEC), which are known to be transfected with difficulties, with transfection efficiencies of over 80%. Furthermore, this was associated with a low cellular toxicity 125.

The possibility of directing the transfection towards a specific area using an external magnetic field gives this approach many advantages for in vivo use 124,125,127. Combining drugs with nanoparticles would reduce systemic side effects and yield a higher concentration of the drug at the target site 127. Administration of a lower amount of the drug would also probably be sufficient (as for the in vitro situation), further reducing systemic side effects. Experiments of delivering anti tumour agents in this way have already been performed successfully in humans 128.