Defining P. falciparum resistance to artemisinin-based combination therapies

恶性疟原虫对青蒿素联合疗法的耐药性的定义

• 批准号: 9319626
• 负责人: David A Fidock
• 金额: $ 37.9万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9319626
• 关键词: Address; Admixture; Alleles; Amodiaquine; Anopheles Genus; Anti-malarial drug resistance; Antimalarials; Artemisinins; Binding; Biological Assay; Blood; Cambodia; Cambodian; Candidate Disease Gene; Chemicals; Chloroquine; Clinical; Combination Drug Therapy; Combined Modality Therapy; Complement; Complex Genetic Trait; Containment; Culicidae; Drug resistance; Erythrocytes; Event; Exposure to; Failure; Falciparum Malaria; Genes; Genetic; Genetic Crosses; Heme; Hepatocyte; Human; In Vitro; Inheritance Patterns; Injectable; Investigation; Linkage Disequilibrium; Malaria; Measures; Mediating; Mediator of activation protein; Meiotic Recombination; Metabolic Clearance Rate; Methods; Molecular; Monitor; Mosquito Control; Multi-Drug Resistance; Mus; Mutation; Parasite resistance; Parasites; Patient Isolators; Patients; Pattern; Pharmaceutical Preparations; Pharmacodynamics; Phenotype; Plasmodium falciparum; Point Mutation; Population; Predisposition; Process; Quantitative Trait Loci; Recombinants; Recording of previous events; Reporting; Resistance; Resistance profile; Role; Sequence Analysis; Sporozoites; Structure; Testing; Transfection; Treatment Efficacy; Validation; asexual; base; benflumetol; clinical efficacy; efficacy study; experimental study; field study; genetic approach; genetic variant; genome sequencing; humanized mouse; in vivo; innovation; interdisciplinary approach; molecular marker; mouse model; novel therapeutic intervention; pressure; public health relevance; pyronaridine; resistance gene; resistant strain; sound; success; tool; trait; translational impact; whole genome; zinc finger nuclease

DESCRIPTION (provided by applicant): Artemisinin-based combination therapies (ACTs) have been pivotal in reducing the global burden of Plasmodium falciparum (Pf) malaria. Their clinical efficacy, however, is threatened by the recent emergence in Cambodia of artemisinin (ART) resistance, defined as reduced rates of parasite clearance. Cambodian parasites show a highly differentiated population structure with minimal chromosomal admixture, which, we hypothesize, allows resistant subpopulations to survive ART exposure by maintaining complex genetic traits in states of linkage disequilibrium. In Aim 1 we test the hypothesis that the kelch gene (PF3D7_1343700), very recently reported to be associated with delayed parasite clearance in patient isolates, constitutes a central determinant of emerging ART resistance across Cambodian Pf subpopulations. To test this, we will use kelch-specific zinc finger nucleases (ZFNs) to revert kelch mutations to the wild-type allele in clinically defined resistant parasites, and to introduce the same mutations into sensitive parasites. Kelch-edited clones will be tested for ART resistance using ring-stage survival assays (RSA) that correlate closely with longer clearance half-lives in vivo and that identify the signature trait of ART-induced early ring stage parasite entry into quiescence. In Aim 2 we address the hypothesis that ART resistance is multifactorial and are defined by subpopulation-specific complex genetic traits. To test this we will study patterns of inheritance in Pf genetic crosses between clinically defined ART-resistant isolates (representing each of the three subpopulations KH2-4) and the sensitive NF54 clone. These crosses take advantage of a new humanized mouse model that allows Pf sporozoites to develop in engrafted human hepatocytes and be recovered in infused human red blood cells. Recombinant progeny will be subjected to whole-genome sequence (WGS) analysis and their ART susceptibility will be quantified using RSA assays. Quantitative trait loci analysis will be used to localize the primary chromosomal regions associated with resistance, and candidate genes will be validated using ZFN-based gene editing. These studies are expected to quantify the role of kelch and define subpopulation-specific secondary determinants. In Aim 3 we address the equally important topic of resistance to the ACT partner drugs, namely lumefantrine, amodiaquine, piperaquine and pyronaridine. Using the humanized mouse model, we will implement genetic crosses with field isolates resistant to amodiaquine or piperaquine, and perform in vitro selection studies for all drugs. WGS analysis will be followed by ZFN-based validation, and mechanistic studies will be implemented to test the hypothesis that Pf partner drug resistance is achieved via reduced drug accumulation and drug-heme binding. Our multidisciplinary approach to defining the genetic and molecular basis of resistance to ACT drugs will provide powerful new investigational tools, and be of direct translational impact in providing markers to readily track resistance and identify appropriate treatment and containment strategies.

描述（由申请人提供）：基于青蒿素的联合疗法（ACT）对于减少全球恶性疟原虫（Pf）疟疾负担至关重要。然而，它们的临床疗效受到柬埔寨最近出现的青蒿素（ART）耐药性（定义为寄生虫清除率降低）的威胁。柬埔寨寄生虫表现出高度分化的群体结构，具有最少的染色体混合，我们假设，这使得抗性亚群体能够通过在连锁不平衡状态下维持复杂的遗传性状而在ART暴露下生存。在目标 1 中，我们检验了以下假设：最近报道称 kelch 基因 (PF3D7_1343700) 与患者分离株中的寄生虫清除延迟有关，该基因构成了柬埔寨 Pf 亚群中出现的 ART 耐药性的核心决定因素。为了测试这一点，我们将使用 kelch 特异性锌指核酸酶 (ZFN) 将 kelch 突变恢复为临床定义的抗性寄生虫中的野生型等位基因，并将相同的突变引入敏感寄生虫中。 Kelch 编辑的克隆将使用环阶段生存测定 (RSA) 进行 ART 抗性测试，该测定与较长的体内清除半衰期密切相关，并识别 ART 诱导的早期环的标志性特征 阶段寄生虫进入静止状态。在目标 2 中，我们提出了这样的假设：ART 耐药性是多因素的，并且由亚群特异性的复杂遗传特征定义。为了测试这一点，我们将研究临床定义的 ART 抗性分离株（代表三个亚群 KH2-4 中的每一个）和敏感 NF54 克隆之间 Pf 遗传杂交的遗传模式。这些杂交利用了一种新的人源化小鼠模型，该模型允许 Pf 子孢子在移植的人肝细胞中发育，并在输注的人红细胞中恢复。重组后代将接受全基因组序列 (WGS) 分析，并使用 RSA 检测量化其 ART 敏感性。数量性状位点分析将用于定位与抗性相关的主要染色体区域，候选基因将使用基于 ZFN 的基因编辑进行验证。这些研究预计将量化 kelch 的作用并定义亚群特异性的次要决定因素。在目标 3 中，我们讨论了同样重要的主题，即对 ACT 伙伴药物（即苯芴醇、阿莫地喹、哌喹和咯萘啶）的耐药性。利用人源化小鼠模型，我们将与对阿莫地喹或哌喹耐药的野外分离株进行基因杂交，并对所有药物进行体外筛选研究。 WGS 分析之后将进行基于 ZFN 的验证，并将实施机制研究来检验 Pf 伴侣耐药性是通过减少药物积累和药物血红素结合来实现的假设。我们定义 ACT 药物耐药性的遗传和分子基础的多学科方法将提供强大的新研究工具，并在提供标记物以轻松追踪耐药性并确定适当的治疗和遏制策略方面具有直接的转化影响。