The high incidence of malaria, coupled to the increasing occurrence of parasite resistance against commonly used anti-malaria drugs and the absence of an effective vaccine, makes the implementation of alternative strategies to combat this killer disease imperative. Antisense technology is one such alternative strategy, which makes use of complementary oligodeoxynucleotides designed to inhibit specific parasite mRNA sequences. These bind to their complementary mRNA target sequences and inhibit their translation. In this study <font face="symbol">a</font>-I-tubulin was chosen as protein target due to its vital role in cell division. Three antisense ODNs (ASL-1, ASL-2 and ASL-3) were designed, complementary to different regions of the parasite á-I-tubulin mRNA. ASL-1 was complementary to the region surrounding the translation initiation codon, with ASL-2 and ASL-3 being complementary to nucleotides 92-118 and 118-144, respectively. These regions were predicted to be accessible for ODN hybridization by RNA secondary structure determination and subsequent analysis. The above ODNs were modified to offer protection against nuclease degradation, by the inclusion of a 15 nucleotide, self-complementary 3’ terminal loop. The stability of these modified ODNs was investigated in serum-containing culture medium, by means of reversed-phase HPLC. These studies showed that the ODN was not significantly degraded after 6 hours of incubation at 37°C in complete culture medium. A sequence-specific inhibition of parasite proliferation of 20% was obtained after antisense ODN, ASL-1 was incubated with ring-phase parasitised cultures. The inclusion of cationic liposomal transfection reagent, DOSPER, abolished inhibition of parasite growth. Only minimal parasite inhibition was observed with ASL-2 and ASL-3. Studies on the effect of parasite phase on ODN uptake showed ODN uptake into trophozoite phase cultures to be 10x higher than in ring-phase cultures. No inhibition was obtained after ASL-1 ODN incubation with trophozoite-phase cultures. Higher uptake in the trophozoite-phase could be explained by modifications introduced into the erythrocyte membrane, by the maturing parasite (72% of trophozoite and only 14.6% of ring-phase parasitised cultures contained ODNs). Retrospective analyses showed that alpha-I-tubulin mRNA was available for ASL-1 hybridization only 40%-50% of the time. The lack of inhibition of the trophozoite developmental stage, by any of the ODNs tested, suggests that the targeted site is no longer available for hybridization. Since ASL-1 was directed to the initiation site and the surrounding sequences it is likely that the ODN is unable to compete with the initiation complex during active translation. The low inhibition observed during the ring phase appears to be due to two factors, low uptake in this phase and availability of the target site prior to translation. Retrospective analysis further showed that the mismatch control ODNs, particularly MSL-1, are complementary to several parasite nucleotide sequences. Inhibition due to antisense ODN ASL-1 could therefore be increased to 33%, when sequence-specific MSL-1 inhibition was not deducted. Inhibitory efficacy of ODNs targeted against the malaria parasite is therefore dependent on the choice of target protein, the local mRNA secondary structure of the target sequence and the transcription and translation stages of the target mRNA and protein, respectively, which include the parasite phases during which these take place.
Thesis (PhD (Biochemistry))--University of Pretoria, 2007.