Understanding Emerging artemisinin partner drug resistance in eastern Africa - ABSTRACT Malaria remains a significant public health challenge, with 234 million cases and 593,000 deaths in Africa in 2021. The emergence of partial resistance to artemisinin in Africa will likely worsen this situation leading to failure to control the parasite with the main frontline drugs artemisinin combination therapies. Arteminsinin resistance also will likely lead to resistance to partner drugs used in combination. Evidence suggests that resistance to lumefantrine, the partner drug in the most common ACT in Africa, artemether-lumefantrine (AL), is also emerging. Our proposal leverages multiple parasite lines from UK travelers who failed AL therapy, including isolates with high-level stable lumefantrine resistance, to investigate resistance mechanisms. We aim to gain insight and understand the underlying cellular and genetic alterations associated with lumefantrine resistance using multi-omics, murine genetic crosses, and CRISPR technology. We will achieve this through the following specific aims: Specific Aim 1: Conduct a detailed analysis of in vitro phenotypes and genome variation in P. falciparum isolates from African patients who failed AL treatment from the UK research biobank. Using two high-level resistant, intermediate and sensitive parasites, we will clone these parasites by limiting dilution to ensure purity and phenotype them through standard IC50 testing. Targeted sequencing of known drug resistance genes will identify potential mutations, and isolates will be assessed for gametocyte production for use in genetic crosses. This aim will create a well-characterized renewable resource related to lumefantrine as well as artemisinin resistance Specific Aim 2: Determine genomic, transcriptional, and translational differences associated with reduced lumefantrine sensitivity. Using well-defined isolates from the UK Malaria Reference Lab, we will perform telomere to telomere genome sequencing, single-cell RNA sequencing, bulk RNAseq, and proteomics to identify stage-specific expression changes and potential causative mutations. We will use CRISPR gene editing to evaluate the impact of known MDR1 and CRT mutations as well as other discovered candidates on lumefantrine resistance. Specific Aim 3: Identify quantitative trait loci (QTLs) associated with stable in vitro lumefantrine resistance through genetic crosses in humanized mice with human hepatocyte grafts. We will cross lumefantrine-resistant parasites with complementary drug-susceptible parasites, analyze recombinants under drug pressure, and perform QTL mapping. CRISPR editing will validate the polymorphisms associated with lumefantrine susceptibility. This comprehensive investigation will yield a valuable extensive bank of characterized isolates, actionable molecular markers, and insights into the mechanisms of lumefantrine resistance, advancing our understanding and control of malaria lumefantrine resistance.
