| 研究課題/領域番号 |
18K19901
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| 研究機関 | 東京大学 |
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研究代表者 |
Cabral Horacio 東京大学, 大学院工学系研究科(工学部), 准教授 (10533911)
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| 研究分担者 |
内田 智士 東京大学, 大学院工学系研究科(工学部), 特任助教 (20710726)
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| 研究期間 (年度) |
2018-06-29 – 2020-03-31
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| キーワード | mRNA / aptamer / hybridization / polymeric nanocarrier / theophylline |
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| 研究実績の概要 |
Messenger RNA (mRNA) have high potential for developing safe gene-based therapies. Because the in vivo application of free mRNA is limited, carrier systemsust be used for overcoming the stability, safety and delivery issues of mRNA. Such carrier systems should also be able to selectively activate the mRNA in the target cell types for achieving protein translation. Thus, we are developing carriers for triggered mRNA translation based on mRNA/aptamer-polymer assemblies for achieving selective protein expression in targeted tissues after sensing endogenous or exogenous molecules. We have prepared aptamers that bind to theophylline as a model for this study. These aptamers showed similar binding affinity to theophylline to the literature. The aptamers were effectively hybridized to the mRNA by temperature control. Then, PEG was conjugated with theophylline. The PEG-theophylline conjugate showed a binding affinity to the aptamer 100-fold lower than that of theophylline. The PEG-theophylline was associated with the mRNA-aptamer hybrids. These mRNA-aptamer hybrids coated with the polymer were stable in serum containing conditions and showed 1000-fold inhibition of the translation efficacy compared to free mRNA in cell free experiments. The addition of theophylline removed the PEG-theophylline chains from the hybrid, which recovered the protein translation.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We have developed a model mRNA carrier strategy based on mRNA/aptamer-polymeric assemblies for functional and targeted transfection. By using a model system, we will demonstrate the concept in biological settings in vitro and in vivo. We are planning to patent this system.
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| 今後の研究の推進方策 |
During next year, we will determine the effect of the design mRNA/aptamer-polymeric assemblies on the stability, safety and efficacy. We will prepare a series of PEG-theophylline conjugates with different molecular weights. Then, the mRNA/aptamer-polymeric assemblies will be evaluated for protein translation in cell free conditions with and without the addition of theophylline. The assemblies will then be studied in cells by using mRNA encoding luciferase with and without the addition of theophylline. The ability of the assemblies to perform translation in vivo will be evaluated by doing an administration to the lungs and studying the translation capability with and without administration of theophylline. The translation will be determined by excusing the lungs, homogenizing and quantifying the luminescence intensity. In addition, we will prepare aptamers for binding to 8-Oxo-2'-deoxyguanosine (8-oxodg), which is a major product of DNA oxidation and a direct measure of oxidative stress. The 8-oxodg aptamers will be hybdrized to mRNA and coated with PEG-8-oxodg conjugates. The translation of the system will be studied in vitro by cell free experiments with and without the addition of free 8-oxodg. Finally, the translation in cancer cells under oxidative conditions will be studied.
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| 次年度使用額が生じた理由 |
We were able to prepare RNA aptamers by ourselves and in sufficient amounts, thereby, reducing the costs of the experiments. Thus, the consumables this year were less than estimated. Next year, we will use the remaining budget for evaluating the mRNA-aptamer polymer nanoassemblies in vivo. The extra budget will be used for purchasing mice and renilla luciferase for real-time in vivo imaging. Moreover, we will attempt to prepare aptamers targeting 8-oxodg, and use this extra budget for obtaining clean products.
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