Abstract
One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. Specific silencing of essential genes by antisense oliogomers has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. In addition, such an approach could be an important biological tool for studying many genes' function by reverse genetics. Here we present a novel methodology of using peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP); namely, octa-D-lysine via the C-terminus, to allow facile delivery through cell membranes. PNAs added to P. falciparum cultures were found exclusively in infected erythrocytes and were eventually localized in nuclei of the parasites at all stages of intra erythrocytic development. We show that these PNAs specifically down regulated both a stably expressed transgene as well as an endogenous essential gene, which significantly reduced parasites' viability. This study paves the way for a simple approach to silence a variety of P. falciparum genes as means of deciphering their function and potentially to develop highly specific and potent antimalarial agents.
Highlights
Malaria is one of the major infectious diseases influencing human kind today
To enable visualization of the peptide nucleic acids (PNAs) molecules in these initial experiments, they were conjugated to two fluorescent markers: Thiazole orange (TO) dye replacing one nucleotide (A) in the middle of the PNA sequence and thiazole red (TR) at the 3’ (Fig. 1A, Fig S1 & Table 1)
We demonstrated that antisense PNA conjugates with a simple octa-D-lysine cell penetrating peptide (CPP) successfully and down-regulate P. falciparum gene expression
Summary
Malaria is one of the major infectious diseases influencing human kind today. The causative agent of the deadliest form of malaria in humans is the protozoan parasite Plasmodium falciparum. This parasite is estimated to infect 300–600 million people worldwide each year, resulting in 1–3 million deaths, primarily of young children and pregnant women [1]. Parasite populations have developed resistance to almost every drug used to treat malaria, including drugs acting at different stages in the complex life cycle of this parasite [2]. In view of the absence of an effective vaccine and the rapid evolution of drug resistance, new approaches are needed in order to fight the disease
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