Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides.

Abstract

We studied inhibition of growth of the malaria parasite Plasmodium falciparum in in vitro culture using antisense (AS) oligodeoxynucleotides (ODNs) against different target genes. W2 and W2mef strains of drug-resistant parasites were exposed to AS ODNs over 48 hr, and growth was determined by microscopic examination and [3H]hypoxanthine incorporation. At ODN concentrations of 1 ,LM, phosphorothioate (PS) ODNs inhibited growth in a targetindependent manner. However, between 0.5 and 0.005 ,LM, ODNs against dihydrofolate reductase, dihydropteroate synthetase, ribonucleotide reductase, the schizont multigene family, and erythrocyte binding antigen EBA175 significantly inhibited growth compared with a PS AS ODN against human immunodeficiency virus, two AS ODNs containing eight mismatches, or the sense strand controls (P < 0.0001). The IC50 was -0.05 IAM, whereas that for non-sequence-specific controls was 15-fold higher. PS AS ODNs against DNA polymerase ai showed less activity than that for other targets, whereas a single AS ODN against triose-phosphate isomerase did not differ significantly from controls. We conclude that at concentrations below 0.5 ,LM, PS AS ODNs targeted against several malarial genes significantly inhibit growth of drugresistant parasites in a nucleotide sequence-dependent manner. This technology represents an alternative method for identifying malarial genes as potential drug targets.

Inhibition of Plasmodium falciparum Proliferationin Vitro by Ribozymes

“…These controls have randomized arm sequence, but maintain an active catalytic domain. A well characterized antisense deoxyoligonucleotide, DHFR105 (17), targeting the dihydrofolate reductase gene was used as a reference control for this study. Unlike the former oligonucleotides, DHFR105 is a fully phosphorothioated DNA molecule, a conventional design in antisense technology.…”

“…Recent success in delineating a sequence-specific response to antisense therapy in P. falciparum in a range of gene targets (17) shows the potential of this technology.…”

“…We show that simple ribozymes can inhibit growth of P. falciparum in a sequence-dependent manner. Furthermore, using an effective antisense deoxyoligonucleotide directed against the dihydrofolate reductase gene (DHFR105) (17), we demonstrate that the carbamoyl-phosphate synthetase ribozymes can perform as well as and potentially better than DHFR105.…”

“…Earlier views that phosphorothioated deoxyoligonucleotides generally decreased parasite viability in a nonsequence-specific manner (16) recent reports of sequence-specific action at oligonucleotide concentrations below 0.5 M directed against a range of targets (17,18). Our investigations center on another class of inhibitory nucleic acids called ribozymes.…”

“…Parasite growth data were determined either by microscopy alone or using the "corrected value" (parasite cell count multiplied by [ 3 H]hypoxanthine incorporation) (17). Experiments were performed blind, and the "code" was revealed following analysis.…”