Gene-Specific Antisense Oligonucleotide Therapy Targeting AMA1: A Potential Malaria Treatment
-
Haleh Hanifian
,
Nader Shahrokhi
,
Mehdi Nateghpour
,
Afsaneh Motevalli Haghi
,
Mehdi Mohebali
,
Abbas Rahimi Foroushani
,
Sepand Razavi Vakhshourpour
Abstract
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.
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5. Heidari A, Dittrich S, Jelinek T, et al. Genotypes and In vivo resistance of Plasmodium falciparum isolates in an endemic region of Iran. Parasitol Res. 2007;100(3):589-592.
6. Eskandarian AA, Keshavarz H, Basco LK, Mahboudi F. Do mutations in Plasmodium falciparum dihydropteroate synthase and dihydrofolate reductase confer resistance to sulfadoxine-pyrimethamine in Iran? Trans R Soc Trop Med Hyg. 2002;96(1):96-98.
7. World Health Organization. WHO guidelines for malaria. Geneva: World Health Organization; 2025.
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11. Vaughan AM, Kappe SH. Genetically attenuated malaria parasites as vaccines. Expert Rev Vaccines. 2017;16(8):765-767.
12. Batista-Duharte A, Sendra L, Herrero MJ, et al. Progress in the use of antisense oligonucleotides for vaccine improvement. Biomolecules. 2020;10(2):316.
13. Bajan S, Hutvagner G. RNA-based therapeutics: from antisense oligonucleot-ides to miRNAs. Cells. 2020;9(1):137.
14. Wicht KJ, Mok S, Fidock DA. Molecular mechanisms of drug resistance in Plasmodium falciparum malaria. Molecular mechanisms of drug resistance in Plasmodium falciparum malaria. Annu Rev Microbiol. 2020;74:431-454.
15. Su X-z, Lane KD, Xia L, Sá JM, Wellems TE. Plasmodium genomics and genetics: new insights into malaria pathogenesis, drug resistance, epidemiology, and evolution. Clin Microbiol Rev. 2019;32(4):e00019-19.
16. Bennett CF. Therapeutic antisense oligonucleotides are coming of age. Annu Rev Med. 2019;70:307-321.
17. Mansoor M, Melendez AJ. Advances in antisense oligonucleotide development for target identification, validation, and as novel therapeutics. Gene Regul Syst Bio. 2008;2:275-295.
18. Plewes K, Leopold SJ, Kingston HW, Dondorp AM. Malaria: what’s new in the management of malaria? Infect Dis Clin North Am. 2019;33(1):39-60.
19. Gerstenlauer C. Recognition and management of malaria. Nurs Clin North Am. 2019;54(2):245-260.
20. Liffner B, Frölich S, Heinemann GK, et al. PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes. Nat Commun. 2020; 11(1):1411.
21. Mardani A, Keshavarz H, Heidari A, Hajjaran H, Raeisi A, Khorramizadeh MR. Genetic diversity and natural selection at the domain I of apical membrane antigen-1 (AMA-1) of Plasmodium falciparum in isolates from Iran. Exp Parasitol. 2012;130(4):456-462.
22. Heidari A, Keshavarz H, Hajjaran H, et al. Genetic Variation and Selection of Domain I of the Plasmodium vivax Apical Membrane Antigen-1(AMA-1) Gene in Clinical Isolates from Iran. Iran J Parasitol. 2013;8(4):536-544.
23. Hanifian H, Nateghpour M, Motevalli Haghi A, et al. Development and optimizing a simple and cost-effective medium for In vivo culture of Plasmodium berghei-ANKA strain with conserving its infectivity in BALB/c mice. BMC Res Notes. 2022;15(1):56.
24. Silvie O, Franetich J-F, Charrin S, et al. A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites. J Biol Chem. 2004;279(10):9490-9496.
25. Nozaki M, Baba M, Tachibana M, et al. Detection of the rhoptry neck protein complex in Plasmodium sporozoites and its contribution to sporozoite invasion of salivary glands. mSphere. 2020;5(4):e00325-20.
26. Aza-Conde J, Reyes C, Suárez CF, et al. The molecular basis for peptide-based antimalarial vaccine development targeting erythrocyte invasion by P. falciparum. Biochem Biophys Res Commun. 2021;534:86-93.
27. Kale S, Pande V, Singh OP, et al. Genetic diversity in two leading Plasmodium vivax malaria vaccine candidates AMA1 and MSP1₁₉ at three sites in India. PLoS Negl Trop Dis. 2021;15(8):e0009652.
28. Sparvoli D, Lebrun M. Unraveling the elusive rhoptry exocytic mechanism of Apicomplexa. Trends Parasitol. 2021; 37(7):622-637.
29. Moitra P, Alafeef M, Dighe K, Frieman MB, Pan D. Selective naked-eye detection of SARS-CoV-2 mediated by N gene targeted antisense oligonucleotide capped plasmonic nanoparticles. ACS Nano. 2020;14(6):7617-7627.
30. Bennett CF, Krainer AR, Cleveland DW. Antisense oligonucleotide therapies for neurodegenerative diseases. Annu Rev Neurosci. 2019;42:385-406.
31. Kheirandish F, Bandehpour M, Haghighi A, et al. Inhibition of Leishmania major PTR1 gene expression by antisense in Escherichia coli. Iran J Public Health. 2012;41(6):65-71.
32. Crater AK, Manni E, Ananvoranich S. Utilization of inherent miRNAs in functional analyses of Toxoplasma gondii genes. J Microbiol Methods. 2015;108:92-102.
33. Stock RP, Olvera A, Sanchez R, et al. Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers. Nat Biotechnol. 2001;19(3):231-234.
34. Duffy PE, Kaslow DC. A novel malaria protein, Pfs28, and Pfs25 are genetically linked and synergistic as falciparum malaria transmission-blocking vaccines. Infect Immun. 1997;65(3):1109-1113.
35. Epp C, Li F, Howitt CA, et al. Chromatin associated sense and antisense noncoding RNAs are transcribed from the var gene family of virulence genes of the malaria parasite Plasmodium falciparum. RNA. 2009;15(1):116-127.
36. Barker RH, Metelev V, Rapaport E, Zamecnik P. Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1996;93(1):514-518.
37. Kolevzon N, Nasereddin A, Naik S, Yavin E, Dzikowski R. Use of peptide nucleic acids to manipulate gene expression in the malaria parasite Plasmodium falciparum. PLoS One. 2014;9(1):e86802.
38. Razavi Vakhshourpour S, Nateghpour M, Shahrokhi N, et al. Potential of RH5 antisense on Plasmodium falciparum proliferation abatement. Iran J Parasitol. 2022;17(4):525-534.
39. Noonpakdee W, Pothikasikorn J, Nimitsantiwong W, Wilairat P. Inhibition of Plasmodium falciparum proliferation In vivo by antisense oligodeoxynucleotides against malarial topoisomerase II. Biochem Biophys Res Commun. 2003;302(4):659-664.
40. Lee DH, Chu KB, Kang HJ, et al. Protection induced by malaria virus-like particles containing codon-optimized AMA-1 of Plasmodium berghei. Malar J. 2019; 18(1):394.
2. Heidari A, Keshavarz H. The drug resistance of Plasmodium falciparum and P. vivax in Iran: a review article. Iran J Parasitol. 2021;16(2):173-185.
3. World Health Organization. Guidelines for the treatment of malaria. 3rd ed. Geneva: WHO; 2015. https://iris.who.int/bitstream/handle/10665/162441/9789241549127_eng.pdf
4. Heidari A, Keshavarz H, Shojaee S, Raeisi A, Dittrich S. In vivo susceptibility of Plasmodium vivax to chloroquine in Southeastern Iran. Iran J Parasitol. 2012;7(2):8-14.
5. Heidari A, Dittrich S, Jelinek T, et al. Genotypes and In vivo resistance of Plasmodium falciparum isolates in an endemic region of Iran. Parasitol Res. 2007;100(3):589-592.
6. Eskandarian AA, Keshavarz H, Basco LK, Mahboudi F. Do mutations in Plasmodium falciparum dihydropteroate synthase and dihydrofolate reductase confer resistance to sulfadoxine-pyrimethamine in Iran? Trans R Soc Trop Med Hyg. 2002;96(1):96-98.
7. World Health Organization. WHO guidelines for malaria. Geneva: World Health Organization; 2025.
8. Kaslow DC. Malaria vaccine research & innovation: the intersection of IA2030 and zero malaria. NPJ Vaccines. 2020;5:109.
9. Miahipour A, Keshavarz H, Heidari A, et al. Genetic variation of MSP-1 gene in Plasmodium vivax isolated from patients in Hormozgan Province, Iran using SSCP-PCR. Iran J Parasitol. 2012;7(4):1-7.
10. Mansouri S, Heidari A, Keshavarz H, Fallah P, Bairami A, Mahmoudi E. Genetic diversity of merozoite surface protein-5 (MSP-5) of Plasmodium vivax isolates from malaria patients in Iran. BMC Infect Dis. 2023;23(1):807.
11. Vaughan AM, Kappe SH. Genetically attenuated malaria parasites as vaccines. Expert Rev Vaccines. 2017;16(8):765-767.
12. Batista-Duharte A, Sendra L, Herrero MJ, et al. Progress in the use of antisense oligonucleotides for vaccine improvement. Biomolecules. 2020;10(2):316.
13. Bajan S, Hutvagner G. RNA-based therapeutics: from antisense oligonucleot-ides to miRNAs. Cells. 2020;9(1):137.
14. Wicht KJ, Mok S, Fidock DA. Molecular mechanisms of drug resistance in Plasmodium falciparum malaria. Molecular mechanisms of drug resistance in Plasmodium falciparum malaria. Annu Rev Microbiol. 2020;74:431-454.
15. Su X-z, Lane KD, Xia L, Sá JM, Wellems TE. Plasmodium genomics and genetics: new insights into malaria pathogenesis, drug resistance, epidemiology, and evolution. Clin Microbiol Rev. 2019;32(4):e00019-19.
16. Bennett CF. Therapeutic antisense oligonucleotides are coming of age. Annu Rev Med. 2019;70:307-321.
17. Mansoor M, Melendez AJ. Advances in antisense oligonucleotide development for target identification, validation, and as novel therapeutics. Gene Regul Syst Bio. 2008;2:275-295.
18. Plewes K, Leopold SJ, Kingston HW, Dondorp AM. Malaria: what’s new in the management of malaria? Infect Dis Clin North Am. 2019;33(1):39-60.
19. Gerstenlauer C. Recognition and management of malaria. Nurs Clin North Am. 2019;54(2):245-260.
20. Liffner B, Frölich S, Heinemann GK, et al. PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes. Nat Commun. 2020; 11(1):1411.
21. Mardani A, Keshavarz H, Heidari A, Hajjaran H, Raeisi A, Khorramizadeh MR. Genetic diversity and natural selection at the domain I of apical membrane antigen-1 (AMA-1) of Plasmodium falciparum in isolates from Iran. Exp Parasitol. 2012;130(4):456-462.
22. Heidari A, Keshavarz H, Hajjaran H, et al. Genetic Variation and Selection of Domain I of the Plasmodium vivax Apical Membrane Antigen-1(AMA-1) Gene in Clinical Isolates from Iran. Iran J Parasitol. 2013;8(4):536-544.
23. Hanifian H, Nateghpour M, Motevalli Haghi A, et al. Development and optimizing a simple and cost-effective medium for In vivo culture of Plasmodium berghei-ANKA strain with conserving its infectivity in BALB/c mice. BMC Res Notes. 2022;15(1):56.
24. Silvie O, Franetich J-F, Charrin S, et al. A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites. J Biol Chem. 2004;279(10):9490-9496.
25. Nozaki M, Baba M, Tachibana M, et al. Detection of the rhoptry neck protein complex in Plasmodium sporozoites and its contribution to sporozoite invasion of salivary glands. mSphere. 2020;5(4):e00325-20.
26. Aza-Conde J, Reyes C, Suárez CF, et al. The molecular basis for peptide-based antimalarial vaccine development targeting erythrocyte invasion by P. falciparum. Biochem Biophys Res Commun. 2021;534:86-93.
27. Kale S, Pande V, Singh OP, et al. Genetic diversity in two leading Plasmodium vivax malaria vaccine candidates AMA1 and MSP1₁₉ at three sites in India. PLoS Negl Trop Dis. 2021;15(8):e0009652.
28. Sparvoli D, Lebrun M. Unraveling the elusive rhoptry exocytic mechanism of Apicomplexa. Trends Parasitol. 2021; 37(7):622-637.
29. Moitra P, Alafeef M, Dighe K, Frieman MB, Pan D. Selective naked-eye detection of SARS-CoV-2 mediated by N gene targeted antisense oligonucleotide capped plasmonic nanoparticles. ACS Nano. 2020;14(6):7617-7627.
30. Bennett CF, Krainer AR, Cleveland DW. Antisense oligonucleotide therapies for neurodegenerative diseases. Annu Rev Neurosci. 2019;42:385-406.
31. Kheirandish F, Bandehpour M, Haghighi A, et al. Inhibition of Leishmania major PTR1 gene expression by antisense in Escherichia coli. Iran J Public Health. 2012;41(6):65-71.
32. Crater AK, Manni E, Ananvoranich S. Utilization of inherent miRNAs in functional analyses of Toxoplasma gondii genes. J Microbiol Methods. 2015;108:92-102.
33. Stock RP, Olvera A, Sanchez R, et al. Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers. Nat Biotechnol. 2001;19(3):231-234.
34. Duffy PE, Kaslow DC. A novel malaria protein, Pfs28, and Pfs25 are genetically linked and synergistic as falciparum malaria transmission-blocking vaccines. Infect Immun. 1997;65(3):1109-1113.
35. Epp C, Li F, Howitt CA, et al. Chromatin associated sense and antisense noncoding RNAs are transcribed from the var gene family of virulence genes of the malaria parasite Plasmodium falciparum. RNA. 2009;15(1):116-127.
36. Barker RH, Metelev V, Rapaport E, Zamecnik P. Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1996;93(1):514-518.
37. Kolevzon N, Nasereddin A, Naik S, Yavin E, Dzikowski R. Use of peptide nucleic acids to manipulate gene expression in the malaria parasite Plasmodium falciparum. PLoS One. 2014;9(1):e86802.
38. Razavi Vakhshourpour S, Nateghpour M, Shahrokhi N, et al. Potential of RH5 antisense on Plasmodium falciparum proliferation abatement. Iran J Parasitol. 2022;17(4):525-534.
39. Noonpakdee W, Pothikasikorn J, Nimitsantiwong W, Wilairat P. Inhibition of Plasmodium falciparum proliferation In vivo by antisense oligodeoxynucleotides against malarial topoisomerase II. Biochem Biophys Res Commun. 2003;302(4):659-664.
40. Lee DH, Chu KB, Kang HJ, et al. Protection induced by malaria virus-like particles containing codon-optimized AMA-1 of Plasmodium berghei. Malar J. 2019; 18(1):394.
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| Issue | Vol 21 No 3 (2026) | |
| Section | Original Article(s) | |
| Keywords | ||
| Plasmodium berghei Malaria 2′-O-methyl Antisense oligonucleotide | ||
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How to Cite
1.
Hanifian H, Shahrokhi N, Nateghpour M, Motevalli Haghi A, Mohebali M, Rahimi Foroushani A, Razavi Vakhshourpour S. Gene-Specific Antisense Oligonucleotide Therapy Targeting AMA1: A Potential Malaria Treatment. Iran J Parasitol. 2026;21(3):370-380.
