Agents that produce their effects through an antisense mechanism offer the possibility of developing highly specific alternatives to traditional pharmacological antagonists, thereby providing a novel class of therapeutic agents, ones which act at the level of gene expression. Among the antisense compounds, antisense RNA produced intracellularly by an expression vector has been used extensively in the past several years. This review considers the advantages of the antisense RNA approach over the use of antisense oligodeoxynucleotides, the different means by which one may deliver and produce antisense RNA inside cells, and the experimental criteria one should use to ascertain whether the antisense RNA is acting through a true antisense mechanism. Its major emphasis is on exploring the potential therapeutic use of antisense RNA in several areas of medicine. For example, in the field of oncology antisense RNA has been used to inhibit several different target proteins, such as growth factors, growth factor receptors, proteins responsible for the invasive potential of tumor cells and proteins directly involved in cell cycle progression. In particular, a detailed discussion is presented on the possibility of selectively inhibiting the growth of tumor cells by using antisense RNA expression vectors directed to the individual calmodulin transcripts. Detailed consideration is also provided on the development and potential therapeutic applications of antisense RNA vectors targeted to the D2 dopamine receptor subtype. Studies are also summarized in which antisense RNA has been used to develop more effective therapies for infections with certain viruses such as the human immunodeficiency virus and the virus of hepatitis B, and data are reviewed suggesting new approaches to reduce elevated blood pressure using antisense RNA directed to proteins and receptors from the renin-angiotensin system. Finally, we outline some of the problems which the studies so far have yielded and some outstanding questions which remain to be answered in order to develop further antisense RNA vectors as therapeutic agents.
The influence of age on the rate of recovery of alpha 1- and alpha 2-adrenergic receptors was determined in rat brain following their irreversible inhibition by phenoxybenzamine (PBZ). Fischer 344 rats (3- or 24-months old) were administered two doses of vehicle or PBZ at 12-hour intervals. At various times after the last dose of PBZ, alpha-adrenergic receptors were quantified in washed membranes of cerebral cortex and hypothalamus, using [3H]prazosin and [3H]rauwolscine to label alpha 1- and alpha 2-adrenergic receptors, respectively. Recovery of prazosin binding sites in cortex and hypothalamus and of rauwolscine binding sites in cortex was significantly delayed in aged versus young rats. Administration of [3H]PBZ to young and aged rats revealed no apparent differences in the concentration or rate of disappearance of PBZ or its metabolites that could account for these age-related changes. These data suggest that the synthesis of both alpha 1- and alpha 2-adrenergic receptors is impaired in brain tissue from aged rats.
To determine the uptake and distribution of oligodeoxynucleotides in brain, a 20-mer phosphorothioated oligodeoxynucleotide complementary to a portion of the D2 dopamine receptor mRNA was fluorescently labeled with fluorescein isothiocyanate (FITC) and injected into the lateral cerebral ventricles of mice. At various survival times after the injection, the brains were removed, fixed, sectioned, and viewed under a fluorescent microscope. The results showed that the oligodeoxynucleotide was rapidly taken up into the brain. Initially the label was relatively diffusely spread throughout the interstitial spaces of the brain, then became redistributed to the cellular compartments. The signal extended from those forebrain nuclei located immediately in contact with the ventricles, such as the corpus striatum, septum, and hippocampus, to areas further removed from the ventricles, such as the cerebral cortex, nucleus accumbens, and substantia nigra. When the FITC-labeled D2 antisense oligodeoxynucleotide was given once daily for 4 d, the signal intensity seen 24 h after the last injection appeared to be of greater intensity overall compared to that seen after a single injection. At early time-points the oligodeoxynucleotide signals appeared to be punctuated and were found in cell bodies as well as in proximal dendritic processes. However, not all cells were equally labeled, suggesting an uneven uptake and accumulation of the D2 antisense into the various cell types. At later time-points the fluorescent signal appeared granular; at these times the injected material was largely degraded. These studies show that a D2 dopamine receptor antisense oligodeoxynucleotide is rapidly taken up from cerebral ventricles into brain, becomes widely distributed throughout the brain tissue to areas far removed from direct contact with the ventricles, and appears to accumulate to a different extent in the various brain areas and cell types.
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