DEALING WITH ANTIBIOTIC RESISTANCE--ANTISENSE TECHNOLOGY

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DESCRIPTION (Adapted from Applicant's Abstract): Drug resistance is a major obstacle in the conquest of bacterial infections. This proposal targets a critical issue in the treatment of bacterial infectious diseases: the need to develop strategies aimed at preserving the effectiveness of currently available aminoglycoside antibiotics. The investigators focus on resistance to the aminoglycoside amikacin mediated by the aminoglycoside 6'-N-acetyltransferase AAC(6')-Ib, which can also modify several other aminoglycoside antibiotics. The long term goal of this research project is to develop the use of antisense oligonucleotides as tools to selectively inhibit the expression of aac(6')-Ib and other related genes. The specific aims for this grant proposal are: 1a) Identification of synthetic oligonucleotides and analogs that promote cleavage of aac(6')-Ib mRNA by RNase H or RNase P. 1b) Bioavailability studies of synthetic oligonucleotides and oligonucleotide analogues. RNase H or RNase P can mediate inhibition of gene expression by degradation of mRNA in the presence of appropriate antisense molecules. The general strategy of this specific aim will consist of the utilization of two conceptually different approaches to identify regions in the mRNA available for interaction with oligonucleotides. With this information, oligodeoxynucleotides and analogues will be designed to inhibit the expression of aac(6')-Ib by inducing RNase H degradation of the mRNA. The information will also be utilized for the development of antisense oligoribonucleotides that inhibit the expression of aac(6')-Ib by inducing RNase P degradation of the mRNA. Following, the investigators will initiate studies on the cell uptake of oligonucleotides and analogues. 2a) Development of peptide nucleic acid (PNA) molecules that inhibit expression of AAC(6')-Ib. 2b) Bioavailability studies of PNA molecules. A novel strategy to inhibit gene expression using antisense technology is now available with the synthesis of peptide nucleic acids (PNAs). PNAs may be developed as antimicrobial agents in prokaryotic systems. The investigators will test the in vitro and in vivo activity of several PNA molecules to interfere with the expression of aac(6')-Ib. The investigators will then study the bioavailability of naked and liposome encapsulated PNA molecules.