Genetically Specific Therapy Against Pathogenic Bacteria

描述(申请人提供)：随着细菌对广谱抗生素产生抗药性，人类正在输掉与感染细菌的军备竞赛。这既是一个健康问题，也是一个环境问题。健康问题是，我们正在失去有效治疗许多细菌感染的能力。环境问题是，我们正在通过大量使用抗生素来扰乱我们周围的微生物生态，这些抗生素不分青红皂白地杀死细菌。我们需要一种新的策略，它将针对病原体，而不影响共生细菌或人类宿主，并且随着细菌的进化而不断进化，以保持对感染细菌的控制。我们应用的广泛、长期目标是为这一新战略奠定基础。在这种方法中，细菌将被一种合成病毒或类似病毒的颗粒感染，这种病毒将反义RNA带入细菌细胞，而不是杀死多种细菌的广谱抗生素。读者可能知道，RNA在细胞中的几个用途之一是调节基因的表达。我们将模仿大自然，专门设计RNA，只敲除致病细菌中的关键基因，而对有益细菌或人类宿主都没有影响。这种类似病毒的颗粒将携带几种不同的反义RNA，每一种都足以杀死或使目标特定的致病细菌变得无毒。细菌将发现很难进化出对这种方法的抗药性，原因有两个：1)因为反义RNA是针对病原体的基因组设计的，可以随着病原体基因组的变化而重新设计；2)因为细菌将感染多个致命RNA，所以不会有强大的选择压力，有利于能够抵抗任何一种RNA影响的细菌变体。当对任何一种病原体没有选择优势时，细菌对多个致死剂产生抗药性的可能性很小。我们的研究战略依赖于三个专业领域：1)生物信息学，从相关基因组数据中提取所有可能击倒病原体关键基因的反义RNA序列；2)纳米科学，诱导病毒样颗粒从组成蛋白质和核酸中自组装；3)微生物遗传学和生理学，以：a)将人类知识添加到计算机输出中，作为优先考虑潜在靶点的指南，以及b)对病原体进行实验，以评估反义RNA的影响。这三个专业领域体现在埃里克·雅各布森(生物信息学)、杰夫·布林克(纳米科学)和斯坦利·马洛伊(微生物学)的合作实验室中。在这个最初的项目中，我们选择了沙门氏菌作为目标。沙门氏菌是一种重要的病原菌，其遗传和毒力机制已被广泛研究，这将是解释实验数据的一大优势。此外，沙门氏菌对几种抗生素产生了抗药性。如果我们在沙门氏菌的实验室中取得成功，我们的策略应该适用于各种传染病。

DESCRIPTION (provided by applicant): Humans are losing the arms race against infectious bacteria as bacteria evolve resistance to broad-spectrum antibiotics. This is both a health problem and an environmental problem. The health problem is that we are losing the ability to effectively treat many bacterial infections. The environmental problem is that we are disrupting the microbial ecology around us by intensive use of antibiotics that kill bacteria indiscriminately. We need a new strategy that will target pathogens without affecting either commensal bacteria or the human host, and that will evolve continually as bacteria evolve to maintain control over infectious bacteria. The broad, long term objective of our application is to lay the foundations for such a new strategy. In this approach, instead of a broad spectrum antibiotic that kills many types of bacteria the bacteria would be infected by a synthetic virus, or virus-like particle, that brings into the bacterial cells antisense RNA. As the reader probably knows, one of the several uses of RNA in the cell is to regulate the expression of genes. We will emulate nature by designing RNA specifically to knock down critical genes in pathogenic bacteria only, and to have no effect on either beneficial bacteria or the human host. The virus-like particles will carry several different antisense RNA's, each of which separately will be sufficient to either kill or render nonvirulent the particular pathogenic bacterium that is being targeted. Bacteria will find it very difficult to evolve resistance to this approach for two fundamental reasons: 1) Because the antisense RNA's are designed to be specific to the pathogen's genome, they can be redesigned as the pathogen's genome changes, and 2) because the bacteria will be infected with multiple lethal RNA, there will not be a strong selection pressure favoring bacterial variants that can resist the effects of any one of the RNA's. The chances of a bacterium developing resistance to multiple lethal agents, when there is no selection advantage for developing resistance to any single agent, is small. Our research strategy rests on three areas of expertise: 1) bioinformatics to extract from the relevant genomic data all the antisense RNA sequences that could potentially knock down critical genes in the pathogenic bacteria, 2) nanoscience to induce the self-assembly of the virus-like particles from the constituent proteins and nucleic acids, and 3) microbial genetics and physiology to: a) add human knowledge to the computer outputs as a guide to prioritizing potential targets and b) to do the experiments on the pathogens in order to assess the effects of the antisense RNA. These three areas of expertise are embodied in the collaborating laboratories of Eric Jakobsson (bioinformatics), Jeff Brinker (nanoscience) and Stanley Maloy (microbiology). For this initial project we have chosen Salmonella as the target. Salmonella is an important pathogen whose genetics and mechanisms of virulence have been extensively studied, which will be a big advantage in interpreting experimental data. Also, Salmonella is acquiring resistance to several antibiotics. If we succeed in the laboratory with Salmonella, our strategy should be applicable to a wide variety of infectious diseases.