2018 Fiscal Year Annual Research Report
Development of a novel nanotubes gene delivery system to suppress invasion process of Babesia
| Project/Area Number |
18F18091
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| Research Institution | Obihiro University of Agriculture and Veterinary Medicine |
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Principal Investigator |
五十嵐 郁男 帯広畜産大学, 原虫病研究センター, 教授 (80159582)
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| Co-Investigator(Kenkyū-buntansha) |
RIZK MOHAMED 帯広畜産大学, 原虫病研究センター, 外国人特別研究員
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| Project Period (FY) |
2018-04-25 – 2020-03-31
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| Keywords | Piroplasm / siRNA / Glycophorin / Nanoparticles / Gene therapy |
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| Outline of Annual Research Achievements |
1) Synthesis of siRNA and nanostructure forming dipeptides: siRNA and Phenylalanine-dehydrophenylalanine (PA-DHPA) were synthesized and purchased from Cambridge Research Biochemicals (Cleveland, UK) and Ambion- ThermoFisher (Tokyo, Japan), respectively.
2) In vitro siRNA protection assay: An electrophoretic mobility and releasing assays of siRNA- loaded Phenylalanine-dehydrophenylalanine (PA-DHPA) nanoparticles formulations were conducted in fetal bovine serum (FBS) for different periods of time. The results demonstrated that 5 mg PA-DHPA, when used in a constant ratio (1:1) with siRNA, provides an optimal condition for the protection and the highest release of siRNA in presence of serum.
3) Hemolytic assay for PA-DHPA was performed and the results revealed the none hemolytic effect of PA-DHPA.
4) In vitro Piroplasm growth inhibition assay: The inhibitory effects of cattle or equine glycophorin C siRNA loaded PA-DHPA on piroplasm growth were evaluated and significant inhibitions of the in vitro growth of piroplasm parasites were observed after treatment by glycophorin C siRNA from an initial parasitemia of 1 %.
5) Effect of PA-DHPA on the erythrocytes on uninfected mice: The effect of PA-DHPA on mice was evaluated using 8-week-old BALB/c mice. Non-significant changes (P>0.05) were observed in the body weight and hematology profiles between the healthy control mice and PA-DHPA- treated mice.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The research plan is a little bit delayed than the schedule due to the synthesis process of Glycophorin C siRNA and Phenylalanine-dihydroxyphenylalanine (PA-DHPA) was taken a long time from the company.
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| Strategy for Future Research Activity |
1. Growth inhibition assay: I will design and evaluate the in vitro inhibitory effects of different types of Glycophorin either A or B siRNA against the growth of different piroplasm parasites.
2. Merozoite invasion assay: Merozoite invasion assay will be performed to determine the efficacy of siRNA loaded GP-DNPs in the silencing of glycophorin receptors into RBCs.
3. Chemotherapeutic evaluation in mice : I will design and evaluate the inhibitory effects of different types of Glycophorin either A, B or C siRNA against the growth of B. microti in mice.
4. Reverse transcription PCR assay and Western blotting: The expression of glycophorin receptors will be determined in the RNA and total proteins extracted piroplasm parasites treated with GP-DNPs- using RT-PCR assay and Western blotting.
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