A novel antisense therapeutic for treatment of Aspergillus fumigatus infections

描述(由申请人提供)：Guild建议开发一种基于反义寡聚体的抗真菌药物，有效对抗人类病原体烟曲霉。由烟曲霉引起的侵袭性曲霉病(IA)死亡率高达40%-60%，现已超过侵袭性念珠菌病成为最常见的死亡原因，尤其是在免疫功能低下的患者中。众所周知，目前的抗真菌药物很难成功治疗IA，因为临床疗效不佳，耐药菌株的出现也越来越多。尽管迫切需要新的治疗策略，但在超过12年的时间里，还没有开发出针对烟曲霉菌的新的抗真菌药物。因此，迫切需要一种新的方法来开发新的抗曲霉靶向策略。磷酸二脂吗啉低聚物(PMOS)是一种人工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进行碱基配对，产生空间位阻的翻译机制。PMOS已被作为抗菌药物进行了研究，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(CPPs)结合的PMOS，这些CPPs可以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，在细胞应激反应中起重要作用。我们团队的研究已经证实，钙调神经磷酸酶是烟曲霉菌生长所必需的，它的缺失会产生许多非致命性缺陷。抑制钙调神经磷酸酶可阻止侵袭性真菌疾病。作为概念验证，将开发针对烟曲霉钙调神经磷酸酶A(CNAA)基因的CPP-PMO，并通过以下特定目的在体外测试其对途径的干扰和生长抑制。目的1.寻找一种能在烟曲霉菌体内最佳蓄积、对人体细胞毒性最小的穿透肽。将文献中的一组CPP与针对白色念珠菌制定的CPP进行比较。顶端积累的多肽将通过竞争性靶向多肽文库进行优化，然后在相关的人类细胞系中进行初步的脱靶毒性测试。目的2.证明利用CPP-PMO构建成功击倒烟曲霉菌CNAA。将针对CNAA基因的起始点设计一个PMO，并将其连接到目标1的CPP上。CPP-PMO将在培养的烟曲霉菌上进行测试，终点为CNAA蛋白敲除、下游效应调控、生长抑制和脱靶毒性。CNAA抑制药FK506将作为阳性的CNAA抑制对照。影响：该项目将生产一种CPP-PMO，扰乱烟曲霉菌的生长。在第二阶段的项目中，我们将在免疫抑制的IA小鼠模型中单独和联合现有的抗真菌药物来评估CPP-PMOS对CNAA和其他基因靶点的作用。这一新的方法针对一个关键的、经过充分验证的烟曲霉菌毒力因子，将对未来IA的治疗模式产生重大影响。

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.