CIRCUITO 1 DE LA LÍNEA SANTA ROSA – EPICLACHIMA 46 kV (CASO

Antisense ODNs are short stretches of synthetic RNA, typically 10 to 30 bases long. Sequences of this length are likely to be unique to the target gene and are known to be taken up by cells. Shorter sequences may lack sequence specificity while longer sequences may not be taken up by cells so easily and also suffer from specificity breakdown due to looping out of non-hybridising sequences (Kregnow et al. 1995).

The ideal anti sense drug should be stable in the intracellular and extracellular environment, be able to cross cell membranes, demonstrate hybridisation specificity, and have a low non-sequence related toxicity (Stein et al. 1993). The first two o f these properties are related to the biochemical properties of the ODN and can be biochemically modified. The last two are a function of the antisense sequence and its ability to interact specifically with intracellular molecules.

Unmodified ODNs contain pbospbodiester bonds which are readily digested by 5 ’ or 3’ exonucleases as well as endonucleases (Tidd 1990). These enzymes are present in both extracellular and intracellular fluids restricting their use in-vivo, although they can be used for in-vitro experiments utilising serum free media. A number of strategies may be used to increase their resistance to degradation by nucleases. The first generation of modified ODNs contained modified pbospbodiester bridges between nucleotides. These were created either by utilising the methylphosphonate or phosphorothioate derivatives (Figure 1.9), changing the glycosidic linkage from the p to the a anomeric form or capping or altering the 3 ’ or 5’ ends o f the oligomer molecule. Second generation modifications have included the synthesis o f phoshorothioate-phosphodiester co-polymers possessing some o f the properties o f both modified and unmodified ODNs (Ghosh et al. 1993). Alternatively, the peptide nucleic acid analogue (PNA) has the entire deoxyribose phosphate backbone exchanged for a chemically different, but structurally homorphous, polyamide backbone composed o f (2-aminoehyl) glycine units (Frank-Kamenetskij 1991). These derivatives can bind to single-stranded genomic

Base HO — P BB O Base Base

L

Figure 1.7: Chemical structure of oligonucleotide derivatives

DNA by Watson-Crick base pairing after displacing the other DNA strand, and to double stranded genomic DNA by forming a triple helix in the major groove. However, these compounds do not have the ability to enter living cells.

Some o f the properties of unmodified phosphodiester and unmodified methylphosphonate and phosphorothioate ODN are shown in Table 1.5. The phosphorothioate derivative has the best combination o f properties and has been the most commonly used type o f ODN for experimental purposes. This derivative has therefore been utilised for the experiments presented in this thesis and in those of the previous RAFT fellows in this project.

Property Phosphodiester Methylphosphonate Phosphorothioate

Biologic stability - + + + + +

Cellular Uptake + + + +

Hybridisation + + + + + +

RNase H activation + + + - + + +

Table 1.8 Properties of normal and modified oligodeoxynucleotides Note: - p oor, + fa ir, + + good, + + + excellent.

ODNs do not readily enter cells as, with the exception o f methylphosphonate derivatives, ODNs are polyanionic (Kregnow et al. 1995). They are therefore similar to most antineoplastic agents which are o f low molecular weight, hydrophilic, and incapable o f passively diffusing across cell membranes. Cellular uptake o f ODNs has been found to be dependent upon time, concentration, energy and temperature which are characteristic o f an active process (Crooke 1991). These findings led to the discovery that ODNs are taken up by cells by two active processes: fluid phase endocytosis and receptor-mediated endocytosis. Once across the membrane, the mechanisms by which they are distributed throughout the cell are controversial. Fluorescent-labelled ODN microinjected into the cytoplasm of cells are found to rapidly accumulate in the nucleus (Leonetti et al. 1991). However,

when fluorescent-labelled ODNs are placed in tissue culture media, the labelled molecules accumulate in vacuoles, presumably in endosomes and lysosomes, within the cytoplasm forming a punctate, perinuclear pattern (Cerruzi et al. 1990). In contrast to ODN localisation when the compounds are introduced by microinjection, there is no visible fluorescence in the nucleus itself, suggesting that the release of ODNs from these vacuoles is an inefficient process. Methylphosphonate derivatives, however, are uncharged particles that have been reported to enter cells via passive diffusion (Miller 1991).

Since ODN uptake is inefficient, several different strategies have been employed to augment this process in order to increase efficiency. ODNs have been conjugated with synthetic polypeptides such as poly-L-lysine, cholesterol, and transferrin (Leonetti et al. 1993). In addition, liposomes containing ODNs can be targeted to reach cell surface determinants with appropriate monoclonal antibodies (Leonetti et al. 1993). The poly-L-lysine modification may mask the negative charge on the ODN, therefore destabilizing the endosomal membranes and permitting ODN escape from the endosomal compartment. Conjugation with cholesterol permits the binding o f ODN to apolipoprotein E and to low-density lipoprotein (LDL) which allows internalisation o f the ODN via the LDL receptor (Leonetti et al. 1993). Cationic lipids such as N-[l(-2,3-dioeyloxy)propyl]-n,n]n-trimethylammonium chloride (DOTMA, Lipofectin®) and some antifiingal drugs such as amphotericin B have also been shown to increase oligomer uptake by cells (Bennett et al. 1992). Lipofectin® not only increases the cellular uptake of ODN, but also increases the uptake o f ODN into the nucleus.