Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 21 3 2026 09 02 370 380 Haleh Hanifian Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Nader Shahrokhi Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran Mehdi Nateghpour Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Afsaneh Motevalli Haghi Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Mehdi Mohebali Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Abbas Rahimi Foroushani Department of Biostatistics and Epidemiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Sepand Razavi Vakhshourpour Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2025 08 02 2026 09 01 Background: We evaluated the effects of 2′-O-methyl (2′-OMe) antisense oligonucleotides targeting the apical membrane antigen 1 (AMA1) gene of Plasmodium berghei using In vivo and In vivo models. Methods: This study was conducted from 2019 to 2022 in Tehran, Iran. Antisense gapmer oligonucleotides targeting conserved regions of the AMA1 gene were designed using Vector NTI, mfold, and sfold and synthesized with 2′-OMe modifications. P. berghei ANKA cultures were treated with various ASO concentrations, and parasitemia was assessed by Giemsa-stained smears and real-time PCR. In vivo efficacy was evaluated in BALB/c mice infected intraperitoneally with P. berghei and treated subcutaneously with ASOs or control oligonucleotides; parasitemia was monitored microscopically. Results: Both antisense oligonucleotides showed low cytotoxicity, with cell viability exceeding 89% at the tested concentrations. In vitro, both antisense oligonucleotides significantly reduced parasitemia compared with the sense control (P=0.01), with no significant difference between the two antisense sequences (P>0.05). Both oligonucleotides also reduced AMA1 expression, with the 183–202 oligonucleotide showing the greatest inhibitory effect. In vivo, the AMA1 183–202-treated group showed significantly lower parasitemia on day 2 post-infection (P=0.048), while lower parasitemia was observed in the antisense-treated groups on subsequent assessment days. Conclusion: 2′-O-methyl antisense oligonucleotides targeting AMA1 reduced parasite growth and AMA1 expression In vivo and were associated with reduced parasitemia in P. berghei-infected mice. These preliminary findings support further investigation of AMA1-directed antisense therapy as a potential gene-specific antimalarial strategy. Larger, adequately powered studies are required to confirm efficacy and evaluate ASO delivery, stability, safety, and durability of gene silencing. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/4708 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/4708/1512