Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis.

Abstract

We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes-the intracellular stage of the parasite-in murine macrophages in culture. After 24 hr of incubation with 10 pM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites.The life cycle of Leishmania involves two successive hosts: the sandfly, in which the parasite grows as a free promastigote, and the mammals, where the amastigote stage multiplies in a parasitophorous vacuole ofthe host's macrophages. These protozoan parasites display a particular process of RNA trans-splicing leading to the presence of a 39-nucleotide-long sequence, termed mini-exon, at the 5' end of all mRNAs (1). This sequence originates in an 89-nucleotidelong mini-exon derived pre-RNA (medRNA) (2).This mini-exon sequence offered an excellent target for antisense oligonucleotides (3). Indeed, this allowed the demonstration of the general occurrence of trans-splicing in Leishmania and trypanosomes (4, 5). Oligonucleotides complementary to the mini-exon sequence were shown to inhibit Trypanosoma brucei and Leishmania enriettii mRNA translation in cell-free assays, in a sequence-dependent manner (6-8). It was also shown that an anti-mini-exon 9-mer, covalently linked to an acridine derivative, was able to kill the procyclic forms of T. brucei in culture (9).For cellular or in vivo purposes, chemically modified oligonucleotides are of interest (10). In particular, modifications can be introduced in the molecules to make them resistant to nucleases (9, 11). Phosphorothioate (PS) analogues that are resistant to DNases and that mediate the degradation of the target RNA by RNase H are receiving particular attention for use in cultured cells and in vivo (12,13).We investigated the effect of PS antisense oligonucleotides on the development of the protozoan parasite Leishmania amazonensis, which is the causal agent of a cutaneous disease in South America. We targeted either the mini-exon sequence or the intron part of the medRNA of the parasite. We report the successful control of the development of L. amazonensis in culture by PS oligonucleotides. MATERIALS AND METHODSOligonucleotide Synthesis and Analysis. Unmodified oligodeoxynucleotides...

A Lipid-modified Phosphoinositide-specific Phospholipase C (TcPI-PLC) Is Involved in Differentiation of Trypomastigotes to Amastigotes of Trypanosoma cruzi

“…Although antisense RNA oligonucleotides have been used extensively to reduce the expression of target genes in mammalian cells (26,27), their use in trypanosomatids has been limited to the targeting of the mini-exon sequence in Leishmania amazonensis as a form of chemotherapy (37), and to reduce the expression of a surface glycoprotein in T. cruzi (38). Our results demonstrate that their use to reduce the expression of functional enzymes in T. cruzi is feasible and that the technique could therefore have wider application when other techniques, such as gene targeting disruption, are not feasible.…”

Targeted Reduction in Expression ofTrypanosoma cruziSurface Glycoprotein gp90 Increases Parasite Infectivity

“…Using such an approach, antiparasitic activities of antisense oligonucleotides towards obligatory intracellular protozoans have been reported. By treating macrophages infected with Leishmania amazonensis for 24 h with phosphorothioate oligonucleotide targeted to the miniexon sequence present at the 5Ј end of every parasite mRNA, cure of about 30% of infected macrophages was observed (14). Rapaport et al (16) demonstrated that the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum is a good target for sequence-dependent inhibition of plasmodial growth by exogenously administered phosphorothioate oligonucleotides.…”

Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides.

“…In vitro, AS ODNs have been shown to inhibit replication of both Trypanosoma brucei (6) and Leishmania amazonensis (7). In vivo, AS ODNs have been used to cure ducks of hepatitis B…”