Defining P. falciparum resistance to artemisinin-based combination therapies

描述（由申请人提供）：以青蒿素为基础的联合疗法（ACTs）在减轻恶性疟原虫（Pf）疟疾的全球负担方面发挥了关键作用。然而，它们的临床疗效受到柬埔寨最近出现的青蒿素耐药性（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种群结构，染色体混合最少，我们假设，这使得抗性亚种群通过在连锁不平衡状态下保持复杂的遗传性状，在抗逆转录病毒药物暴露中存活下来。在目的1中，我们检验了kelch基因（PF3D7_1343700）的假设，该基因最近被报道与患者分离物中寄生虫清除延迟有关，是柬埔寨Pf亚群中出现的抗逆转录病毒耐药性的核心决定因素。为了验证这一点，我们将使用kelch特异性锌指核酸酶（ZFNs）将临床定义的耐药寄生虫中的kelch突变还原为野生型等位基因，并将相同的突变引入敏感寄生虫。kelch编辑的克隆将使用环期生存测定法（RSA）测试抗逆转录病毒耐药性，这与体内较长的清除半衰期密切相关，并确定抗逆转录病毒诱导的早期环的特征

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.