The purpose of this proposal is to exploit the potential for gene-specific activities of synthetic oligonucleotides (ODNs) in an animal model involving drug metabolism. The mechanistic actions of nuclease resistant ODNs include sequence-specific interactions with nucleic acids as antigene or antisense molecules or with transcriptional regulatory proteins in a manner that mimics the genomic cis-elements resulting in the modulation of gene expression. The investigators intend to test the hypothesis that optimal oligonucleotide structures can be identified that modulate the expression of cytochrome P450 isoforms in vivo. Further, that gene expression modulation will provide insights into the regulation of these genes' expression and their reliance on endogenous substrates and extrahepatic expression. In vivo studies are proposed because ODN entry into and distribution within the cell is not equivalent between studies conducted in cultured cells and that observed in intact animals.
The specific aims of these studies are to: 1) identify optimal oligonucleotide structures for antisense, antigene, ribozyme and transcriptional regulation of gene expression in vivo, 2) evaluate the mechanism of action of these modulators of gene expression, 3) identify in in vivo modulators a broad spectrum of phase I drug metabolizing enzymes including rat CYP1A1, CYP2B1, CYP2B2, CYP2C11, CYP2E1, CYP3A2 and CYP4A1 and 4) evaluate these gene expression modulators in context dependent expression created by sex hormones, circadian rhythm, xenobiotic exposure and extrahepatic organs. The advantages of this model system are that 1) constitutive gene expression is monitored in the absence of disease, 2) the in vivo efficacy is confirmed by in vitro analysis of enzyme activities and protein levels directly link the target mRNA with observed phenotype, 3) toxicity can be evaluated concomitantly with efficacy, 4) the approach is cost effective, 5) this approach avoids discrepancies that are in cell culture and 6) direct comparison of potency, efficacy and toxicity can be made with linear phosphorothioate ODNs. Future studies will involve a more detailed investigation of the role of cytochrome P450 expression in the regulation of radical oxygen sensitive genes in vivo.
