Oligonucleotides, antisense

Antisense oligonucleotides directed against PKC II led to loss of proliferative capacity in K562 cells (Murray et al., 1993). Dowmnodulation of PKC by antisense had no effect on the proliferation and saturation density in K562 cells (Murray et al., 1997). However, Spitaler et al. (1999) found that down-modulation of PKC with antisense oligonucleotides in HeLa cells led to the induction of apoptosis. 

A selective method of preventing the expression of adhesion molecules or cytokines is the use of antisense oligonucleotides. These oligonucleotides are short sequences of nucleic acids complementary to mRNA sequences of specific proteins of interest. If delivered to the cytoplasmic compartment of cells these oligonucleotides are able to form a complex with their target mRNA. In this way the translation of mRNA into protein by ribosomes is inhibited. The subsequent mRNA degradation by RNAse H results in reduced expression of the protein (see also Chapter 5 for a description of antisense ohgonucleotides as therapeutic modalities). 

ICAM-1, VCAM-1 and E-selectin synthesis was successfully blocked in vitro using these types of molecules [113]. In vivo, the systemic administration of ICAM-1 antisense oligonucleotide prevented and reversed murine colitis without serious side-effects [114]. In a place-bo-controUed trial of the human analogue, the antisense oligonucleotide was effective and well tolerated [115]. 

Local and systemic administration of an NFkB p65 subunit antisense phophorothioate oligonucleotide effectively inhibited experimental cohtis in mice [116]. 

It is important to realize however that these antisense molecules were not specifically targeted to the endothehum. Consequently, the contribution of the endothelial cells to the effects observed is unknown. Furthermore, in these studies adequate control experiments with mismatched ohgonucleotides are essential, since polyanionic agents such as antisense oligonucleotides can exert a broad range of non-specific antisense effects due to non-specific binding to proteins [117]. 

7 Vascular Endothelium in Inflamed Tissue as a Target for Site Selective Delivery of Drugs 

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A newer, highly experimental approach to anxiety therapy is the use of antisense oligonucleotides to the anxiogenic peptide, NPY (44). 

An effective therapeutic agent must also have the abiUty to reach its target sequence m vivo. BioavailabiUty requires that the antisense oligonucleotide be able to pass through the cell membrane, and that it have a low affinity for nontarget cellular compartments and, in animal systems, nontarget organs. 

Cell membranes are lipophilic and designed to be barriers against large anionic molecules, although there is a natural mechanism for intercellular transport of anionic oligonucleotides. In order to enhance membrane transport, antisense oligonucleotides are frequentiy modified by covalent attachment of carrier molecules or lipophilic groups. 

VEGF inhibitors Humanized neutralizing antibodies, antisense oligonucleotides, siRNA, aptamers... 

Oblimersen sodium is a DNA antisense oligonucleotide designed to specifically bind to human bcl-2 mRNA, resulting in catalytic degradation of bcl-2. This results in decreased translation of the protein Bcl-2, which is a cellular antiapoptotic protein. Thus, oblimersen enhances sensitivity to chemotherapy by shifting the intracellular balance to a state in which the cells are more likely to be killed by apoptosis. Currently, it is used in combination chemotherapy for treating advanced melanoma. 

Antisense Oligonucleotides. Figure 1 Schematic representation of the action of antisense oligonucleotides. They bind to their respective target mRNA preventing protein translation. 

Antisense Oligonucleotides. Table 1 Malignant disorders as potential targets for ribozyme gene therapy... 

ASON stands for antisense oligonucleotides. Antisense Oligonucleotides... 

CpG stands for cytosine phosphate guanine dinucleotide in a particular sequence context. CpG motifs are responsible for proliferative effects of antisense oligonucleotides, particularly with respect to B-lymphocytes. Die optimal immune-stimulatory consensus sequence surrounding CpG is R1R2CGY1Y2, where R1 is a purine (mild preference for G), R2 is a purine or T (preference for A), and Y1 and Y2 are pyrimidines (preference for T). 

The antisense oligonucleotide LErafAON against the serine/threonine kinase c-Raf has been tested in phase I clinical trials. The antisense oligonucleotides ISIS-5132, which also inhibits c-Raf, and ISIS-3521, which inhibits PKC, went through different phase clinical trials with solid tumour patients. Unfortunately, no objective responses occurred with these PKI. GEM-231, an oligonucleotide targeting the RIa subunit of protein kinase A is currently undergoing phase I/II clinical trials alone or in combination with traditional therapy for the treatment of solid cancers [3]. 

AP 12009 Antisense oligonucleotide TGF- 32 Glioma, pancreatic carcinoma, melanoma... 

Wahlestedt C, Salmi P, Good L, Kela J, Johnsson T, Hokfelt T, Broberger C, Porreca F, Lai J, Ren K, Ossipov M, Koshkin A, Jakobsen N, Skouv J, Oerum H, Jacobsen MH, Wengel J (2000) Potent and nontoxic antisense oligonucleotides containing locked nucleic acids, Proc Natl Acad Sd USA 97 5633-5638... 

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