A novel antisense therapeutic for treatment of Aspergillus fumigatus infections

一种治疗烟曲霉感染的新型反义疗法

基本信息

批准号：
8714774
负责人：
Augustine Anthony DiNovo
金额：
$ 17.01万
依托单位：
GUILD ASSOCIATES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8714774
关键词：
Acute Animal Model Antifungal Agents Antifungal Therapy Antisense RNA Aspergillosis Aspergillus Aspergillus fumigatus Bacteria Base Pairing Biochemical Biotechnology Calcineurin Calcineurin inhibitor Candida Candida albicans Candidiasis Cause of Death Cell Wall Cells Cellular Stress Characteristics Clinical Collection Cyclosporine Databases Defect Disease Drug resistance Effectiveness Ensure Enzymes Essential Genes Evaluation FK506 Fungal Drug Resistance Future Gene Targeting Genes Goals Growth High Pressure Liquid Chromatography Human Human Cell Line Immunocompromised Host Immunosuppressive Agents In Vitro Infection Label Lead Literature Measurable Measures Mediating Messenger RNA Modeling Molds Mus Mutation Mycoses Pathway interactions Peptide Library Peptides Performance Pharmaceutical Preparations Phase Protein phosphatase Proteins Proteomics Research Serum Site Sushi Domain Testing Therapeutic Time Toxic effect Toxicity Tests Treatment Efficacy Virulence Virulence Factors Western Blotting Work antimicrobial antisense nucleic acid base biological adaptation to stress calcineurin phosphatase clinical efficacy design fungus immunosuppressed in vitro testing knock-down member mortality new technology novel novel strategies novel therapeutics nucleic acid analog pathogen phosphorodiamidate morpholino oligomer public health relevance resistant strain

项目摘要

DESCRIPTION (provided by applicant): Guild proposes to develop an antisense oligomer-based antifungal effective against the human pathogen Aspergillus fumigatus. With a 40%-60% mortality rate, invasive aspergillosis (IA) due to A. fumigatus now surpasses invasive candidiasis as the most frequent fungal cause of death, especially amongst immunocompromised patients. Successful therapy for IA with current antifungals is notoriously difficult due to poor clinical efficacy and the increasing emergence of drug resistant strains. Despite this acute need for novel therapeutic strategies, there has not been a new class of antifungals targeting A. fumigatus developed in over 12 years. Therefore, a novel approach is urgently needed to develop new anti-Aspergillus targeting strategies. Phosphorodiamidate morpholino oligomers (PMOs), synthetic uncharged analogs of nucleic acids, are an antisense biotechnology that functions by base pairing with target gene mRNA, producing steric blockade of the translational machinery. PMOs have been investigated as antimicrobials against bacteria, but applications to fungal pathogens are rare. Our team has recently developed PMOs conjugated to cell penetrating peptides (CPPs) that inhibit the growth of the fungal pathogen Candida albicans. Calcineurin is a highly conserved protein phosphatase that is important in mediating cell stress responses. Research by our team has established that calcineurin is necessary for A. fumigatus growth, and its deletion produces numerous nonlethal defects. Inhibition of calcineurin halts invasive fungal disease. As proof of concept, a CPP-PMO will be developed against the A. fumigatus calcineurin A (CnaA) gene and tested In Vitro for pathway disruption and growth inhibition through the following specific aims. Aim 1. Identify a cell penetrating peptide that optimally accumulates within A. fumigatus and has minimal toxicity to human cells. A set of CPPs from the literature will be compared to the CPP developed against C. albicans. The top accumulating peptide will be optimized through competitive targeted peptide libraries, and then be tested for preliminary off-target toxicity in relevant human cell lines. Aim 2. Demonstrate successful knockdown of A. fumigatus CnaA using a CPP-PMO construct. A PMO will be designed against the start site of the cnaA gene and be conjugated to the CPP from Aim 1. The CPP- PMO will be tested on cultured A. fumigatus with endpoints of CnaA protein knockdown, downstream effector modulation, growth reduction, and off target toxicity. The CnaA inhibiting drug FK506 will be used as a positive CnaA inhibition control. Impact: This project will produce a CPP-PMO that disrupts A. fumigatus growth. In the Phase 2 project, we will evaluate CPP-PMOs against CnaA and other gene targets both alone and in combination with existing antifungal agents in immunosuppressed murine models of IA. This novel approach targeting a critical well- validated A. fumigatus virulence factor will significantl impact the future treatment paradigm of IA.

描述（由申请人提供）：Guild 提议开发一种有效对抗人类病原体烟曲霉的基于反义寡聚物的抗真菌剂。由烟曲霉引起的侵袭性曲霉病 (IA) 的死亡率为 40%-60%，现已超过侵袭性念珠菌病，成为最常见的真菌死亡原因，尤其是在免疫功能低下的患者中。由于临床疗效不佳和耐药菌株的不断出现，目前的抗真菌药物很难成功治疗 IA。尽管迫切需要新的治疗策略，但 12 年来尚未开发出针对烟曲霉的新型抗真菌药物。因此，迫切需要一种新方法来开发新的抗曲霉靶向策略。磷酸二酰胺吗啉寡聚物 (PMO) 是一种合成的不带电荷的核酸类似物，是一种反义生物技术，通过与靶基因 mRNA 碱基配对发挥作用，产生翻译机制的空间封锁。 PMO 作为细菌抗菌剂已被研究，但应用于真菌病原体的情况很少。我们的团队最近开发了与细胞穿透肽 (CPP) 结合的 PMO，可抑制真菌病原体白色念珠菌的生长。钙调神经磷酸酶是一种高度保守的蛋白磷酸酶，对于介导细胞应激反应非常重要。我们团队的研究已经确定钙调神经磷酸酶对于烟曲霉的生长是必需的，并且其缺失会产生许多非致命缺陷。抑制钙调神经磷酸酶可阻止侵袭性真菌病。作为概念验证，将针对烟曲霉钙调磷酸酶 A (CnaA) 基因开发 CPP-PMO，并通过以下具体目标进行体外途径破坏和生长抑制测试。目标 1. 鉴定一种可在烟曲霉内最佳积累且对人体细胞毒性最小的细胞穿透肽。文献中的一组 CPP 将与针对白色念珠菌开发的 CPP 进行比较。积累最高的肽将通过竞争性靶向肽库进行优化，然后在相关人体细胞系中进行初步脱靶毒性测试。目标 2. 展示使用 CPP-PMO 构建体成功敲低烟曲霉 CnaA。 PMO 将针对 cnaA 基因的起始位点进行设计，并与目标 1 中的 CPP 缀合。 CPP-PMO 将在培养的烟曲霉上进行测试，终点包括 CnaA 蛋白敲低、下游效应子调节、生长减少和脱靶毒性。 CnaA 抑制药物 FK506 将用作阳性 CnaA 抑制对照。影响：该项目将产生扰乱烟曲霉生长的 CPP-PMO。在第二阶段项目中，我们将在 IA 免疫抑制小鼠模型中单独评估 CPP-PMO 对抗 CnaA 和其他基因靶标的效果，以及与现有抗真菌药物联合使用的效果。这种针对关键的、经过充分验证的烟曲霉毒力因子的新方法将显着影响 IA 的未来治疗模式。