Dealing with Antibiotic Resistance: Antisense Technology

描述(由申请人提供)：这项提案针对的是对抗日益严重的抗生素耐药性问题的一个关键问题：寻找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lb型[aac(6’)-lb]，这是一种介导对阿米卡星和其他氨基糖苷类药物耐药性的酶。我们的长期目标是开发反义寡核苷酸作为药物工具，选择性地抑制AAC(6‘)-1b的表达。为了开发两种反义化合物，通过不同的机制抑制AAC(6‘)-Lb的表达，我们设计了特异的目标1和2： 1.鉴定促进核糖核酸酶H介导的aac(6‘)-lb mRNA降解的抗核酸酶寡核苷酸类似物。我们将设计核酸酶抗性类似物，并测试它们介导表型转换为阿米卡星敏感性的能力，并确定其作用机制。 2.核糖核酸酶P介导的寡核苷酸降解AAC(6‘)-1b mRNA的体内研究和系统分析核酸酶抗性寡核苷酸类似物诱导RNase P裂解的能力。我们将设计编码所选寡核苷酸的质粒，并测试它们是否诱导核糖核酸酶P介导的阿米卡星敏感性转换。我们还将对耐核酸酶的寡核苷酸类似物进行系统研究，以确定哪些类似物(如果有的话)不会影响RNaseP介导的RNA切割。 虽然实现特定目标1和2将是开发反义化合物以保持阿米卡星疗效的重要一步，但仍有许多问题需要解决。其中两个问题是：a)确保反义化合物到达细菌细胞质的传递方法非常有限；b)aac(6‘)-lb基因经常在高拷贝数的质粒中发现；因此，大量的基因拷贝可能使其很难完全关闭表达。具体目标3和4是为处理这些问题而设计的： 3.开发能够将寡核苷酸输送到细胞胞浆中的脂质体制剂。我们将测试几种阳离子脂质体包裹的寡核苷酸类似物到达细胞质的能力。内化过程将通过荧光显微镜进行表征。 4.寻找AAC(6‘)-Lb酶的多肽抑制剂。酶抑制剂可以通过抑制合成的任何残留的AAC(6‘)-Lb蛋白的作用而与反义寡核苷酸产生协同作用。将使用噬菌体展示技术搜索多肽抑制剂。

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.