Enhanced activity of antisense phosphorothioate oligos against leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions.
Leishmania, a parasitic protozoan, infects human macrophages, often causing severe morbidity and mortality. The pathogenic form of this parasite, the amastigote, lives inside the acidic phagolysosomes of infected macrophages. In our attempt to develop anti-miniexon phosphorothioate oligodeoxyribonucleotides (S-oligos) as an alternative chemotherapy against Leishmania, we found that intracellular as well as 'axenic' amastigotes were more susceptible to these S-oligos than were the cultured promastigotes. Lower pH (4.5) and elevated temperature (35 degrees) of the medium were among the direct enhancing factors for killing. Addition of the cationic polypeptide poly-l-lysine (PLL) to the growth medium further enhanced the killing effect of the S-oligo at pH 4.5. The enhancement of specific ablation of mRNA expression was directly correlated to the increased leishmanicidal activity of the S-oligo. This was shown by the increased inhibition of luciferase activity expressed in transgenic Leishmania amazonensis promastigotes by anti-miniexon S-oligo or anti-luciferase S-oligo at acidic pHs and in the presence of PLL. The leishmanicidal effects of S-oligos at acidic pH and in the presence of PLL were related to increased uptake of the S-oligos under these conditions. The rate of S-oligo uptake was enhanced up to 15-fold at pH 4.5. The addition of PLL to the assay medium at acidic pH further enhanced the uptake of S-oligo up to 80-fold. RNase H is known to accentuate the antisense action of S-oligos. We found that at an elevated temperature RNase H activity in Leishmania cell extracts increased about 5-fold. Thus, enhanced uptake of S-oligos at the acidic pH of macrophage phagolysosomes and activation of RNase H may explain the efficient killing of the parasite in macrophages, both in tissue culture and in the animal model, by antisense miniexon oligonucleotide/PLL, when targeted directly to the parasite-containing phagolysosomes.
Mishra
M
M
Department of Microbiology, Meharry Medical College, Nashville, TN 37208, USA.
Porter-Kelley
J M
JM
Singh
P K
PK
Bennett
J R
JR
Chaudhuri
G
G
D000818:Animals; D004195:Disease Models, Animal; D004337:Drug Carriers; D016503:Drug Delivery Systems; D004347:Drug Interactions; D004789:Enzyme Activation; D005856:Germ-Free Life / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007891:Leishmania / Q000187:drug effects* / Q000378:metabolism; D007896:Leishmaniasis / Q000188:drug therapy; D008081:Liposomes; D008156:Luciferases / Q000096:biosynthesis / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D020319:Oligodeoxyribonucleotides, Antisense / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D021261:Parasitic Sensitivity Tests; D010588:Phagosomes / Q000187:drug effects / Q000469:parasitology; D011107:Polylysine / Q000494:pharmacology*; D016914:Ribonuclease H / Q000378:metabolism*; D013696:Temperature; D013873:Thionucleotides / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D014162:Transfection
D004337:Drug Carriers; D008081:Liposomes; D020319:Oligodeoxyribonucleotides, Antisense; D013873:Thionucleotides; D011107:Polylysine; D008156:Luciferases; D016914:Ribonuclease H