Dealing with Antibiotic Resistance: Antisense Technology

描述（由申请人提供）：该提案针对对抗日益严重的抗生素耐药性问题的一个关键问题：寻求旨在保持现有抗生素有效性的策略。我们的模型系统是氨基糖苷6 '-N-乙酰转移酶Ib型[AAC（6'）-Ib]，一种介导对阿米卡星和其他氨基糖苷类的抗性的酶。我们的长期目标是开发反义寡核苷酸作为选择性抑制aac（6 '）-lb表达的药理学工具。为了开发两种通过不同机制抑制aac（6 '）-lb表达的反义化合物，我们设计了具体目标1和2： 1.鉴定促进RNA酶H介导的aac（6 '）-lb mRNA降解的核酸酶抗性寡脱氧核苷酸类似物。我们将设计核酸酶抗性类似物，并测试它们介导表型转化为阿米卡星敏感性的能力，并确定其作用机制。 2. RNA酶P介导的aac（6 '）-lb mRNA被寡核糖核苷酸降解的体内研究和核酸酶抗性寡核糖核苷酸类似物诱导RNA酶P切割的能力的系统分析。我们将设计编码选定寡核苷酸的质粒，并测试它们是否诱导RNase P介导的阿米卡星敏感性转化。我们还将对核酸酶抗性寡核糖核苷酸类似物进行系统研究，以确定哪些（如果有的话）不会损害RNase P介导的RNA切割。 虽然实现具体目标1和2将是开发反义化合物以保持阿米卡星功效的重要一步，但仍有许多问题有待解决。其中两个问题是：a）确保反义化合物到达细菌细胞的细胞质的递送方法非常有限;和B）aac（6 ′）-lb基因通常存在于高拷贝数质粒中;因此，大量的基因拷贝可能使得完全关闭表达非常困难。为解决这些问题，制定了具体目标3和4： 3.开发能够将寡核苷酸递送到细胞胞质溶胶中的脂质体制剂。我们将测试阳离子脂质体包封的寡核苷酸类似物的几种制剂到达细胞质的能力。内化过程将通过荧光显微镜表征。 4.搜索AAC（6 '）-lb酶的肽抑制剂。酶抑制剂可通过抑制合成的任何残余AAC（6 '）-lb蛋白的作用而与反义寡核苷酸具有协同活性。将使用噬菌体展示搜索肽抑制剂。

DESCRIPTION (provided by applicant): This proposal targets a critical issue in the fight against the growing problem of antibiotic resistance: the search for strategies aimed at preserving the effectiveness of currently available antibiotics. Our model system is the aminoglycoside 6'-N-acetyltransferase type lb [AAC(6')-lb], an enzyme that mediates resistance to amikacin and other aminoglycosides. Our long term goal is to develop antisense oligonucleotides as pharmacological tools to selectively inhibit the expression of aac(6')-lb. To develop two kinds of antisense compounds that inhibit expression of aac(6')-lb by different mechanisms we designed specific aims 1 and 2: 1. Identification of nuclease-resistant oligodeoxynucleotide analogs that promote RNase H-mediated degradation of aac(6')-lb mRNA. We will design nuclease-resistant analogs and test their ability to mediate phenotypic conversion to amikacin susceptibility and determine the mechanism of action. 2. In vivo studies on RNase P-mediated degradation of aac(6')-lb mRNA by oligoribonucleotides and systematic analysis of the ability of nuclease-resistant oligoribonucleotide analogs to induce RNase P cleavage. We will design plasmids that code for selected oligoribonucleotides and test if they induce RNase P-mediated conversion to amikacin susceptibility. We will also carry out a systematic study on nuclease-resistant oligoribonucleotide analogs to determine which ones, if any, do not compromise RNase P-mediated cleavage of RNA. While achieving specific aims 1 and 2 will be an important step towards developing antisense compounds to preserve the efficacy of amikacin, many problems will remain to be solved. Two of these problems are: a) delivery methods to insure that antisense compounds reach the bacterial cell's cytoplasm are very limited; and b) the aac(6')-lb gene is often found in high copy number plasmids; as a consequence the large number of gene copies may make it very difficult to completely turn off expression. Specific aims 3 and 4 have been designed to deal with these problems: 3. Development of liposome formulations capable of delivering oligonucleotides into the cell's cytosol. We will test the ability of several formulations of cationic liposome-encapsulated oligonucleotide analogs to reach the cytoplasm. The process of internalization will be characterized by fluorescence microscopy. 4. Search for peptide inhibitors of the AAC(6')-lb enzyme. Enzyme inhibitors could have a synergistic activity with antisense oligonucleotides by inhibiting the action of any residual AAC(6')-lb protein synthesized. Peptide inhibitors will be searched using phage display.