Reducing Nephrotoxicity while enabling read through of missense stop codons by Gentamicin congeners

降低肾毒性，同时能够通过庆大霉素同系物读取错义终止密码子

• 批准号: 9898260
• 负责人: ROBERT L BACALLAO
• 金额: --
• 依托单位: RLR VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898260
• 关键词: Acute; Amino Acids; Aminoglycoside Antibiotics; Aminoglycosides; Antibiotics; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Chronic Kidney Failure; Clinical; Clinical Research; Cultured Cells; Cystic Fibrosis; Data; Defect; Disease; Dose; Dose-Rate; Duchenne muscular dystrophy; Dystrophin; Effectiveness; Elderly; Fluorescence Resonance Energy Transfer; Future; Genes; Gentamicins; Heart Diseases; Histology; Human; Immunohistochemistry; Infection; Injections; Kidney; Kidney Diseases; Label; Lead; Liver diseases; Malignant - descriptor; Malignant Neoplasms; Measures; Mediating; Mendelian disorder; Methods; Modeling; Molecular; Muscular Dystrophies; Nonsense Codon; Nonsense Mutation; Normal Cell; Oncology; Organism; Patients; Plasmids; Production; Protein Biosynthesis; Proteins; Proximal Kidney Tubules; Rattus; Reading Frames; Signal Transduction; Solid Neoplasm; TP53 gene; Terminator Codon; Testing; Therapeutic; Tissues; Transgenic Mice; Translations; Western Blotting; bactericide; base; cell transformation; cell type; design; fluorophore; gene product; human disease; imaging modality; imaging probe; in vivo; insight; interest; intravital imaging; mdx mouse; mouse model; nephrotoxicity; novel; ototoxicity; premature; prevent; ratiometric; response; two photon microscopy

Aminoglycoside antibiotics remain first line therapy for gram negative infections and are used in conjunction with other antibiotics for certain gram positive organisms. Aminoglycosides, especially gentamicin, have also been used to suppress premature stop codon termination of protein synthesis leading to monogenic diseases like cystic fibrosis and Duchene's Muscular Dystrophy. Unfortunately, they still have an unacceptably high rate of dose and duration-dependent nephrotoxicity, especially in the elderly and in patients with chronic kidney, heart and liver disease. We have previously identified non-nephrotoxic, yet bactericidal, congeners of gentamicin that we now propose to use to determine their efficacy in suppressing premature termination codons (PTC). To do this we will use a human proximal tubule cell line with a novel FRET probe allowing for ratiometric quantitation of readthrough of different embedded stop codons. This will be followed by in vivo studies, again using the FRET probe, but now with 2-photon microscopy in kidney proximal tubule cells labeled by subcapsular delivery of specifically designed plasmids based on the cell culture data. To test the clinical potential of this approach we will study the mdx mouse and quantify dystropin synthesis in response to dose and duration dependent therapy. Finally, we will develop a transgenic mouse for the most appropriate stop codon, determined by the rat studies, and quantify readthrough in multiple tissues throughout the body. This is particularly important as many solid tumor cancers have monogenic premature termination codons that lead to malignant transformation of the cell type and these are also responsive to suppression therapy by aminoglycosides. We hypothesize that gentamicin congeners, with markedly reduced nephrotoxicity, will provide enhanced suppression of PTC leading to synthesis of functional proteins needed to reduce and/or prevent multiple PTC diseases. The nontoxic congeners will allow us to directly compare and contrast differences between toxic and nontoxic forms thereby leading to greater understanding of the factors mediating premature termination codon suppression in multiple cell types. These studies will provide the information necessary to enhance translation into clinical studies of multiple diseases.

氨基糖苷类抗生素仍然是革兰氏阴性感染的一线治疗药物，并联合使用 与其他抗生素一起用于某些革兰氏阳性细菌。氨基糖苷类，特别是庆大霉素，也有 被用于抑制导致单基因疾病的蛋白质合成的过早终止密码子 比如囊性纤维症和杜氏肌营养不良症。不幸的是，他们仍然有一个令人无法接受的高比率 剂量和时间依赖性的肾毒性，特别是在老年人和慢性肾脏病患者中， 心脏和肝脏疾病。我们之前已经鉴定出无肾毒性，但具有杀菌作用的同系物 我们现在建议使用庆大霉素来确定它们抑制早产的有效性 密码子(PTC)。为了做到这一点，我们将使用具有新型FRET探针的人类近端小管细胞系，以允许 不同嵌入终止密码子通读的比率定量。这之后将在体内进行。 研究，再次使用FRET探针，但现在用双光子显微镜在肾脏近端小管细胞标记 根据细胞培养数据，通过包膜下传递专门设计的质粒。为了测试临床 这种方法的潜力我们将研究mdx小鼠并量化dystroins的合成与剂量的关系。 和持续时间依赖疗法。最后，我们将开发出一种转基因小鼠，以达到最合适的目的 密码子，由大鼠研究确定，并量化在全身多个组织中的阅读。这是 尤其重要的是，许多实体肿瘤的单基因提前终止密码子会导致 细胞类型的恶性转化，这些细胞也对抑制治疗有反应 氨基糖苷类。我们假设庆大霉素的同系物，肾毒性显著降低， 将提供对PTC的增强抑制，导致合成 减少和/或预防多种PTC疾病。无毒的同系物将使我们能够直接比较 并对比有毒和无毒形式之间的差异，从而更好地理解 在多种细胞类型中调节提前终止密码子抑制的因素。这些研究将提供 加强对多种疾病临床研究的转化所需的信息。