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

基本信息

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
项目类别：
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

项目摘要

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）在减轻疟原虫（PF）疟疾的全球负担方面至关重要。然而，它们的临床功效受到阿秒毒素（ART）耐药性最近出现的威胁，这被定义为寄生虫清除率降低。柬埔寨寄生虫显示出高度分化的种群结构，并具有最小的染色体混合物，我们假设这允许耐药的亚群来通过在连锁不平衡状态下维持复杂的遗传特征来生存ART暴露。在AIM 1中，我们检验了kelch基因（PF3D7_1343700）的假设，最近据报道与患者分离株中延迟的寄生虫清除率有关，构成了柬埔寨PF亚群体中新兴艺术耐药性的中心决定因素。为了测试这一点，我们将使用Kelch特异性锌指核酸酶（ZFN）在临床定义的抗性寄生虫中恢复为野生型等位基因的Kelch突变，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法（RSA）测试与较长的间隙半衰期在体内紧密相关的ART抗性，并确定了艺术诱导的早期环的标志性特征阶段寄生虫进入静止。在AIM 2中，我们解决了以下假设：ART抗性是多因素的，并且由亚群特异性复杂遗传特征定义。为了测试这一点，我们将研究临床定义的耐药分离株（代表三个亚群KH2-4中的每个）和敏感的NF54克隆之间的PF遗传杂交中的遗传模式。这些杂交利用了一种新的人源化小鼠模型，该模型允许PF孢子虫在植入的人肝细胞中发展，并在注入的人类红细胞中回收。重组后代将进行全基因组序列（WGS）分析，其ART敏感性将使用RSA分析进行量化。定量性状基因座分析将用于定位与抗药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。这些研究有望量化Kelch的作用并定义亚群特异性二级决定因素。在AIM 3中，我们谈到了对ACT伴侣药物的抵抗力，即Lumefantrine，Amodiaquine，piperaquine和pyronaridine。使用人源化小鼠模型，我们将用耐氨二喹或果皮的田间分离株实施遗传杂交，并对所有药物进行体外选择研究。 WGS分析将在基于ZFN的验证之后进行，并将实施机械研究以检验以下假设：PF伴侣耐药性是通过降低药物积累和药物 - 血红素结合来实现的。我们定义对ACT药物抗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，并具有直接的翻译影响，以提供标志物，以便于追踪阻力并确定适当的治疗方法和遏制策略。