Potential of RH5 Antisense on Plasmodium falciparum Proliferation Abatement
-
Sepand Razavi Vakhshourpour
,
Mehdi Nateghpour
,
Nader Shahrokhi
,
Afsaneh Motevalli Haghi
,
Mehdi Mohebali
,
Haleh Hanifian
Abstract
Background: Infections by Plasmodium falciparum, are becoming increasingly difficult to treat. Therefore, there is an urgent need for novel antimalarial agents’ discovery against infection. In present study, we described a 2’-O-Methyl gapmer phosphorothioate oligonucleotide antisense targeting translation initiation region of 3D7 strain RH5 gene.
Methods: The study was conducted in Pasteur Institute of Iran in 2020. ODNs effects were measured by microscopic examination and real time RT-PCR. For microscopy, microplates were charged with 2’-OMe ODNs at different dilutions. Unsynchronized parasites were added to a total of 0.4 ml (0.4% parasitemia, 5% red blood cells), and slides were prepared. Proportion of infected cells was measured by counting at least 500 red blood cells.
Results: RH5 genes start codon regions selected as conserved region besed on alignment results. Gap-RH5-As which was complementary to sequence surrounding AUG RH5 start codon significantly reduced parasite growth (>90% at 50 nM) compared to sense sequence control (Gap-RH5-Se) (17%), (P<0.001). RH5 transcripts were dramatically reduced after exposed to ODNs at a concentration of 5-500 nM for 48 h.
Conclusion: Gemnosis delivery of a chimeric gapmer PS-ODN with 2’-OMe modifications at both sides had high antisense activity at low concentrations (10-100 nM) and shown a good efficiency to reach to target mRNA in human RBCs. Anti-parasite effect was correlated to reduction of target gene mRNA level. In addition, 2’-OMe ODNs free delivery is an effective way and does not need any carrier molecules or particles.
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2. Dhuri K, Bechtold C, Elias Quijano, et al. Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development. J Clin Med. 2020;9(6): 2004.
3. Tang X, Liu Y, Xie Z, et al. Efficient Gen-eration of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair. Mol Ther Nucleic. 2016;5(11):e396.
4. Batista-Duharte A, Sendra L, Herrero MJ, et al. Progress in Use of Antisense Oligo-nucleotides for Vaccine Improvement. Bi-omolecules. 2020;10(2): 316.
5. Mansoor M, Melendez AJ. Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel rapeutics. Gene Regul Syst Bio. 2008;2: 275–295.
6. Yoo BH, Bochkareva E, Bochkarev A, et al. 2′-O-methyl-modified phosphorothio-ate antisense oligonucleotides have re-duced non-specific effects in vitro. Nucleic Acids Res. 2004;32(6): 2008-16.
7. Monia B, Lesnikn EA, Gonzalez C, et al. Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense in-hibitors of gene expression. J Biol Chem . 1993;268(19): 14514-14522.
8. Song X, Wang X, MaZicai Y, et al. Site-Specific Modification Using 20-Methoxyethyl Group Improves Specificity and Activity of siRNAs. MoL Ther Nucle-ic Acids. 2017;9: 242-250.
9. Barker RH, Valeri JR, Rapaport E, et al. Inhibition of P. falciparum malaria using an-tisense oligodeoxy nucleotides. Proc NatI Acad Sci U S A. 1995;93(1): 514–518.
10. Luganini A, Caposio P, Landolfo S, Gribaudo G. Phosphorothioate-Modified Oligodeoxynucleotides Inhibit Human Cy-tomegalovirus Replication by Blocking Vi-rus Entry. J Antimicrob Chemother. 2008;52(3): 1111-20.
11. Pouny Y, Rapaport D, Mor A, et al. Inter-action of Antimicrobial Dermaseptin and Its Fluorescently Labeled Analogues with Phospholipid Membranes. Biochemistry. 1992;31(49): 12416-23
12. Sharma Sh, Deoliveira RB, Kalantari P, et al. Innate Immune Recognition of an AT-Rich Stem-Loop DNA Motif in P. falcipa-rum Genome. J Immunol. 2011;35(2):194-207.
13. Kuespert S, Heydn R, Peters S, et al. Anti-sense Oligonucleotide in LNA-Gapmer Design Targeting TGFBR2—A Key Single Gene Target for Safe and Effective Inhibi-tion of TGFβ Signaling. Int J Mol Sci. 2020;21(6): 1952.
14. Miller C, Harris E. Antisense Oligonucleo-tides: Treatment Strategies and Cellular In-ternalization. RNA Dis. 2016;3(4): e1393.
15. Cowman AF, Tonkin Ch, Tham W, et al. Molecular Basis of Erythrocyte Invasion by Malaria Parasites. Cell Host Microbe. 2017;22(2): 232-245.
16. Ord R, Rodriguez M, Lobo Ch. Human Malaria invasion ligand RH5 and its prime candidacy in blood-stage malaria vaccine design. Hum Vaccin Immunother. 2015;11(6): 1465–1473.
17. Lopaticki S, Maier A, Thompson J, et al. Reticulocyte and Erythrocyte Binding-Like Proteins Function Cooperatively in Inva-sion of Human Erythrocytes by Malaria Parasites. Infect Immun. 2011;79(3): 1107-17.
18. Beeson J, Drew D, Boyle M, et al. Mero-zoite surface proteins in red blood cell in-vasion, immunity and vaccines against ma-laria. FEMS Microboil Rev. 2016;40(3): 343-72.
19. Santos J, Josling G, Ross Ph, et al. Red Blood Cell Invasion by Malaria Parasite Is Coordinated by PfAP2-I Transcription Factor. Cell Host Microbe. 2017;21(6): 731–741.
20. Aniweh Y, Gao X, Hao P, et al. P. falcipa-rum RH5-Basigin interaction induces changes in cytoskeleton of host RBC. Cell Microboil. 2017;19(9): e12747.
21. Scarpelli P, Pecenin M , Garcia C. Intracel-lular Ca2+ Signaling in Protozoan Para-sites: An Overview with a Focus on Mito-chondria. Int J Mol Biol. 2021;22(1): 469.
22. Galaway R , Drought L, Fala M , et al. P113 is a merozoite surface protein binds N terminus of P. falciparum RH5. Nat Commun. 2017;8: 14333.
23. Bustamantea L, Bartholdsona S, Crosnier C et al. A full-length recombinant P. falcipa-rum PfRH5 protein induces inhibitory anti-bodies are effective across common PfRH5 genetic variants. Vaccine 2013;31(2): 373–379.
24. Trager W, Jenson J. Cultivation of malarial parasites. Methods Cell Biol. 1978;272: 621–622.
25. Murphy SC, Prentice JL, Williamson K, et al. Real-time quantitative reverse transcrip-tion PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials. Am J Trop Med Hyg. 2012;86(3): 383-394.
26. Ouattara A, Tran T, Doumbo S, et al. Ex-tent and Dynamics of Polymorphism in Malaria Vaccine Candidate Plasmodium falci-parum Reticulocyte–Binding Protein Homologue-5 in Kalifabougou, Mali. Am J Trop Med Hyg. 2018;99(1): 43–50.
27. Roberts T, Langer R , Wood M. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 2020;19: 673–694.
28. Noonpakdee W, Pothikasikorn J, Nim-itsantiwong W, Wilairat P. Inhibition of Plasmodium falciparum proliferation in vitro by antisense oligodeoxynucleotides against malarial topoisomerase II. Biochem Bio-phys Res Commun. 2003;302: 659–664.
29. Rapaport E, Misiura K, Agrawal S, Za-mecnik P. Antimalarial activities of oli-godeoxynucleotide phosphorothioates in chloroquine-resistant Plasmodium falciparum. Proc Natl Acad Sci U S A. 1992; 89: 8577–8580.
30. Barker RH, Jr, Metelev V, Coakley A, et al. Plasmodium falciparum: effect of chemical structure on efficacy and specificity of anti-sense oligonucleotides against malaria in vitro. Exp Parasitol. 1998; 88: 51–59.
31. Garg A, Wesolowski D, Alonso D, et al. Targeting protein translation, RNA splic-ing, and degradation by morpholino-based conjugates in Plasmodium falciparum. Proc NatI Acad Sci U S A. 2015;112: 11935-11940.
32. Kolevzon N, Nasereddin A, Naik S, et al. Use of peptide nucleic acids to manipulate gene expression in malaria parasite Plasmo-dium falciparum. PloS One. 2014;9(1): e86802.
33. Foger F, Noonpakdee W, Loretz B, et al. Inhibition of malarial topoisomerase II in Plasmodium falciparum by antisense nanopar-ticles. Int J Pharm. 2006;319: 139–146.
2. Dhuri K, Bechtold C, Elias Quijano, et al. Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development. J Clin Med. 2020;9(6): 2004.
3. Tang X, Liu Y, Xie Z, et al. Efficient Gen-eration of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair. Mol Ther Nucleic. 2016;5(11):e396.
4. Batista-Duharte A, Sendra L, Herrero MJ, et al. Progress in Use of Antisense Oligo-nucleotides for Vaccine Improvement. Bi-omolecules. 2020;10(2): 316.
5. Mansoor M, Melendez AJ. Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel rapeutics. Gene Regul Syst Bio. 2008;2: 275–295.
6. Yoo BH, Bochkareva E, Bochkarev A, et al. 2′-O-methyl-modified phosphorothio-ate antisense oligonucleotides have re-duced non-specific effects in vitro. Nucleic Acids Res. 2004;32(6): 2008-16.
7. Monia B, Lesnikn EA, Gonzalez C, et al. Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense in-hibitors of gene expression. J Biol Chem . 1993;268(19): 14514-14522.
8. Song X, Wang X, MaZicai Y, et al. Site-Specific Modification Using 20-Methoxyethyl Group Improves Specificity and Activity of siRNAs. MoL Ther Nucle-ic Acids. 2017;9: 242-250.
9. Barker RH, Valeri JR, Rapaport E, et al. Inhibition of P. falciparum malaria using an-tisense oligodeoxy nucleotides. Proc NatI Acad Sci U S A. 1995;93(1): 514–518.
10. Luganini A, Caposio P, Landolfo S, Gribaudo G. Phosphorothioate-Modified Oligodeoxynucleotides Inhibit Human Cy-tomegalovirus Replication by Blocking Vi-rus Entry. J Antimicrob Chemother. 2008;52(3): 1111-20.
11. Pouny Y, Rapaport D, Mor A, et al. Inter-action of Antimicrobial Dermaseptin and Its Fluorescently Labeled Analogues with Phospholipid Membranes. Biochemistry. 1992;31(49): 12416-23
12. Sharma Sh, Deoliveira RB, Kalantari P, et al. Innate Immune Recognition of an AT-Rich Stem-Loop DNA Motif in P. falcipa-rum Genome. J Immunol. 2011;35(2):194-207.
13. Kuespert S, Heydn R, Peters S, et al. Anti-sense Oligonucleotide in LNA-Gapmer Design Targeting TGFBR2—A Key Single Gene Target for Safe and Effective Inhibi-tion of TGFβ Signaling. Int J Mol Sci. 2020;21(6): 1952.
14. Miller C, Harris E. Antisense Oligonucleo-tides: Treatment Strategies and Cellular In-ternalization. RNA Dis. 2016;3(4): e1393.
15. Cowman AF, Tonkin Ch, Tham W, et al. Molecular Basis of Erythrocyte Invasion by Malaria Parasites. Cell Host Microbe. 2017;22(2): 232-245.
16. Ord R, Rodriguez M, Lobo Ch. Human Malaria invasion ligand RH5 and its prime candidacy in blood-stage malaria vaccine design. Hum Vaccin Immunother. 2015;11(6): 1465–1473.
17. Lopaticki S, Maier A, Thompson J, et al. Reticulocyte and Erythrocyte Binding-Like Proteins Function Cooperatively in Inva-sion of Human Erythrocytes by Malaria Parasites. Infect Immun. 2011;79(3): 1107-17.
18. Beeson J, Drew D, Boyle M, et al. Mero-zoite surface proteins in red blood cell in-vasion, immunity and vaccines against ma-laria. FEMS Microboil Rev. 2016;40(3): 343-72.
19. Santos J, Josling G, Ross Ph, et al. Red Blood Cell Invasion by Malaria Parasite Is Coordinated by PfAP2-I Transcription Factor. Cell Host Microbe. 2017;21(6): 731–741.
20. Aniweh Y, Gao X, Hao P, et al. P. falcipa-rum RH5-Basigin interaction induces changes in cytoskeleton of host RBC. Cell Microboil. 2017;19(9): e12747.
21. Scarpelli P, Pecenin M , Garcia C. Intracel-lular Ca2+ Signaling in Protozoan Para-sites: An Overview with a Focus on Mito-chondria. Int J Mol Biol. 2021;22(1): 469.
22. Galaway R , Drought L, Fala M , et al. P113 is a merozoite surface protein binds N terminus of P. falciparum RH5. Nat Commun. 2017;8: 14333.
23. Bustamantea L, Bartholdsona S, Crosnier C et al. A full-length recombinant P. falcipa-rum PfRH5 protein induces inhibitory anti-bodies are effective across common PfRH5 genetic variants. Vaccine 2013;31(2): 373–379.
24. Trager W, Jenson J. Cultivation of malarial parasites. Methods Cell Biol. 1978;272: 621–622.
25. Murphy SC, Prentice JL, Williamson K, et al. Real-time quantitative reverse transcrip-tion PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials. Am J Trop Med Hyg. 2012;86(3): 383-394.
26. Ouattara A, Tran T, Doumbo S, et al. Ex-tent and Dynamics of Polymorphism in Malaria Vaccine Candidate Plasmodium falci-parum Reticulocyte–Binding Protein Homologue-5 in Kalifabougou, Mali. Am J Trop Med Hyg. 2018;99(1): 43–50.
27. Roberts T, Langer R , Wood M. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 2020;19: 673–694.
28. Noonpakdee W, Pothikasikorn J, Nim-itsantiwong W, Wilairat P. Inhibition of Plasmodium falciparum proliferation in vitro by antisense oligodeoxynucleotides against malarial topoisomerase II. Biochem Bio-phys Res Commun. 2003;302: 659–664.
29. Rapaport E, Misiura K, Agrawal S, Za-mecnik P. Antimalarial activities of oli-godeoxynucleotide phosphorothioates in chloroquine-resistant Plasmodium falciparum. Proc Natl Acad Sci U S A. 1992; 89: 8577–8580.
30. Barker RH, Jr, Metelev V, Coakley A, et al. Plasmodium falciparum: effect of chemical structure on efficacy and specificity of anti-sense oligonucleotides against malaria in vitro. Exp Parasitol. 1998; 88: 51–59.
31. Garg A, Wesolowski D, Alonso D, et al. Targeting protein translation, RNA splic-ing, and degradation by morpholino-based conjugates in Plasmodium falciparum. Proc NatI Acad Sci U S A. 2015;112: 11935-11940.
32. Kolevzon N, Nasereddin A, Naik S, et al. Use of peptide nucleic acids to manipulate gene expression in malaria parasite Plasmo-dium falciparum. PloS One. 2014;9(1): e86802.
33. Foger F, Noonpakdee W, Loretz B, et al. Inhibition of malarial topoisomerase II in Plasmodium falciparum by antisense nanopar-ticles. Int J Pharm. 2006;319: 139–146.
| Files | ||
| Issue | Vol 17 No 4 (2022) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v17i4.11280 | |
| Keywords | ||
| Antisense Plasmodium falciparum Malaria | ||
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How to Cite
1.
Razavi Vakhshourpour S, Nateghpour M, Shahrokhi N, Motevalli Haghi A, Mohebali M, Hanifian H. Potential of RH5 Antisense on Plasmodium falciparum Proliferation Abatement. Iran J Parasitol. 2022;17(4):525-534.
