CRISPR‐Cas9‐modified pfmdr1 protects Plasmodium falciparum asexual blood stages and gametocytes against a class of piperazine‐containing compounds but potentiates artemisinin‐based combination therapy partner drugs

Abstract

Summary Emerging resistance to first-line antimalarial combination therapies threatens malaria treatment and the global elimination campaign. Improved therapeutic strategies are required to protect existing drugs and enhance treatment efficacy. We report that the piperazine-containing compound ACT-451840 exhibits single-digit nanomolar inhibition of the Plasmodium falciparum asexual blood stages and transmissible gametocyte forms. Genome sequence analyses of in vitro-derived ACT-451840-resistant parasites revealed single nucleotide polymorphisms in pfmdr1, which encodes a digestive vacuole membrane-bound ATP-binding cassette transporter known to alter P. falciparum susceptibility to multiple first-line antimalarials. CRISPR-Cas9 based gene editing confirmed that PfMDR1 point mutations mediated ACT-451840 resistance. Resistant parasites demonstrated increased susceptibility to the clinical drugs lumefantrine, mefloquine, quinine and amodiaquine. Stage V gametocytes harboring Cas9-introduced pfmdr1 mutations also acquired ACT-451840 resistance. These findings reveal that PfMDR1 mutations can impart resistance to compounds active against asexual blood stages and mature gametocytes. Exploiting PfMDR1 resistance mechanisms provides new opportunities for developing disease-relieving and transmission-blocking antimalarials.

Hexahydroquinolines are antimalarial candidates with potent blood-stage and transmission-blocking activity

“…Because HHQ resistance was the highest in lines harboring the Y290F mutation, we employed a previously derived NF54 line 27 that harbored this mutation (NF54 selY290F ) to verify whether Y290F also conferred resistance at the GAM stage. NF54 selY290F early-stage GAMs showed a 40–74-fold IC 50 shift for all HHQs compared to wild-type NF54 (Supplementary Table 7).…”

“…To test whether these point mutations in PfMDR1 were causal for resistance, we implemented a pfmdr1 -specific CRISPR/Cas9 strategy 27 . Transfection of parental Dd2-B2 parasites yielded the PfMDR1 edF1072L and PfMDR1 edS1075I clones that expressed the F1072L and S1075I mutations respectively, observed in both pfmdr1 copies.…”

UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes

“…Briefly, Cas9 was expressed from a pDC2-based human dhfr plasmid, along with a sequence encoding the specific guide RNA. Expression of the latter was driven either by a short T7 promoter (for pfugt ), via co-expression of the T7 RNA polymerase from a pDC2-bsd plasmid, or via a U6 promoter ( pfact ) 51 . A donor template with homology to the target site was also supplied on a pDC2-bsd plasmid, and contained both the desired nucleotide replacement and also silent mutations in the Cas9 cut site to prevent cleavage of the donor or the modified genome.…”

CRISPR/Cas9 mediated sequential editing of genes critical for ookinete motility in Plasmodium yoelii

“…For editing the genome of P. falciparum , the Cas9/sgRNA expression cassette and donor DNA template were delivered into parasites in two separate vectors with each plasmid carrying a different drug resistance gene (selection marker) [11, 13–15]. After electroporation, parasites transfected with the vectors were enriched by selection of two drugs simultaneously [11, 13, 14]. For the rodent malaria parasite P. yoelii , a single vector system was used because limited independent selection markers were available for the parasites.…”