Defining P. falciparum resistance to artemisinin-based combination therapies

描述（由申请人提供）：青蒿素为基础的联合疗法（ACT）在减少全球恶性疟原虫（Pf）疟疾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的威胁，这种耐药性的定义是寄生虫清除率降低。柬埔寨寄生虫表现出高度分化的人口结构与最小的染色体混合物，我们假设，允许耐药亚群生存ART暴露保持复杂的遗传性状的连锁不平衡状态。在目标1中，我们检验了以下假设：最近报道的与患者分离株中寄生虫清除延迟相关的kelch基因（PF3D7_1343700）构成了柬埔寨Pf亚群中出现ART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）在临床定义的耐药寄生虫中将kelch突变恢复为野生型等位基因，并将相同的突变引入敏感寄生虫。Kelch编辑的克隆将使用环阶段存活测定（RSA）测试ART抗性，所述测定与体内较长的清除半衰期密切相关，并鉴定ART诱导的早期环化的特征性性状。 阶段寄生虫进入静止期。在目标2中，我们提出了ART耐药是多因素的，并由亚群特异性复杂遗传性状定义的假设。为了验证这一点，我们将研究临床定义的ART耐药分离株（代表三个亚群KH 2 - 4中的每一个）和敏感NF 54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。将对重组后代进行全基因组序列（WGS）分析，并使用RSA试验定量其ART易感性。定量性状基因座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的作用kelch和定义亚群特定的次要决定因素。在目标3中，我们讨论了同样重要的ACT伙伴药物耐药性问题，即本芴醇、阿莫地喹、哌喹和咯萘啶。使用人源化小鼠模型，我们将与对阿莫地喹或哌喹耐药的田间分离株进行遗传杂交，并对所有药物进行体外选择研究。WGS分析之后将进行基于ZFN的验证，并将进行机制研究以检验Pf伴侣耐药性是通过减少药物蓄积和药物-血红素结合实现的假设。我们的多学科方法来定义耐药ACT药物的遗传和分子基础，将提供强大的新的研究工具，并在提供标记物方面产生直接的翻译影响，以方便地跟踪耐药并确定适当的治疗和遏制策略。

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.