A New Approach: Using Antisense Oligonucleotides to Target AMA1 Gene in Treatment of Malaria

Abstract Background: Resistance among malaria parasites has emerged against the majority of antimalarial drugs. As a result, the development of novel therapeutic strategies has become increasingly critical. The antisense technique, known for its specificity and minimal resistance and toxicity, has attracted considerable interest from researchers in recent years. Antisense oligodeoxynucleotides (ASOs) represent a promising alternative chemotherapeutic approach for inhibiting the synthesis of specific mRNA and its subsequent translation into protein. These molecules consist of single-stranded DNA or RNA that hybridize with a target gene, typically comprising 15 to 25 nucleotides. The agents inhibit gene expression at the RNA level by blocking transcription or translation of target genes. Their high specificity, efficacy, and low toxicity make them promising candidates for chemotherapy. This study developed an antisense oligonucleotide aimed at inhibiting AMA1 gene in Plasmodium berghei through both in vitro and in vivo methods. Methods: In this study, a modified cultivation technique for Plasmodium berghei parasites was developed to evaluate the in vitro effects of oligodeoxynucleotides (ODNs). Additionally, an in vivo assessment of antisense oligonucleotides was performed using BALB/c mice. Analyses of the AMA-1 gene were performed utilizing Vector NTI 10 software, along with the mfold and sfold tools, to determine optimal gene positions for the design of antisense oligonucleotides. Subsequently, a second-generation antisense oligonucleotide (2-O-methyl) was designed in the Gamper format. The results were evaluated through microscopic examination and real-time reverse transcription polymerase chain reaction (RT-PCR) following oligonucleotide treatment. Results: The findings indicate that AMA-1 mRNA in Plasmodium berghei represents a promising target for 2'-O-Methyl oligonucleotides. Importantly, these antisense oligonucleotides are capable of penetrating red blood cells in their unmodified form, a phenomenon known as gymnosis, without the necessity for biological carriers, thereby effectively inhibiting the growth of the parasite. The levels of parasitemia in all groups treated with antisense oligonucleotides were significantly lower than those observed in the group that received sense oligonucleotides, as determined by both microscopic and molecular analyses via RT-PCR. Conclusion: The findings of this study suggest that antisense oligonucleotides may be an effective, non-toxic, and highly specific novel therapeutic strategy for the treatment of Plasmodium berghei and other Plasmodia-related infections.