| 研究実績の概要 |
This project aimed at creation of gene delivery systems for the treatment of liver diseases. We selected liver fibrosis as a disease model. Hepatic stellate cells (HSCs) play a pivotal role in the development of hepatofibrogenesis. Therefore, we decided to design lipid nanoparticles for the selective mRNA delivery to HSCs. To maximize the clinical translatability of these platforms, we attempted ligand-free targeting and employed a novel microfluidic device called Invasive Lipid Nanoparticle Production device (iLiNP), which has been developed previously by our partners in the Faculty of Engineering, Hokkaido University. First, a library of 14 pH-sensitive lipids were screened in vitro to investigate their mRNA delivery efficiency and transfection efficiency to activated HSCs cell line, LX-2. The lipid library demonstrated a molecular diversity in the polar head group that controlled pKa, and the long distal hydrophobic tail that controlled other physico-chemical properties and performance. The names of the lipids were CL1A6, CL1C6, CL1D6, CL1H6, CL4C6, CL4D6, CL4H6, CL4F6, CL4G6, CL7A6, CL7H6, CL15A6, CL15F6, CL15H6. The prepared The LNPs showed acceptable physico-chemical properties. The five top-performing candidates were further enriched via a second round of screening to evaluate their selectivity to HSCs (LX-2) versus hepatocytes (FL83B). Two promising candidates were identified with high efficiency and selectivity for further in vivo evaluation and optimization. Furthermore, the selected candidates showed high tolerability, with cell viability values above 80%.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
We designed the first platform for mRNA delivery to HSCs, which hold promise for clinical applications in the gene therapy of liver fibrosis. We also achieved ligand-free targeting of HSCs for the first time, which will increase the clinical translatability of the system via increasing the stability, and minimizing the production steps and costs. We are communicating with the Hokkaido University Intellectual Property office to file a patent application. We are also considering submission of the results to a high impact factor journal. Although the research fellow faced severe family-related unavoidable conditions last year, he could rapidly resume his performance. In addition to what was originally-planned, we performed an in-depth technical evaluation of the clinical translation of nanomedicines, which served the scope of the current research project aiming at creation of clinically-translatable nanotherapeutics. The key aspects controlling the clinical translatability of nanomedicines were identified. This technical review was highly-impacted and published in the prestigious journal, Advanced Drug Delivery Reviews, impact factor= 15.47 (Younis MA, Tawfeek HM, Abdellatif AAH, Abdel-Aleem JA, Harashima H. Clinical translation of nanomedicines: Challenges, opportunities, and keys. Adv Drug Deliv Rev. 2022 Feb;181:114083. doi: 10.1016/j.addr.2021.114083). Moreover, the fellow has effectively contributed to a second high impact review on the in vivo fate of nanocarriers and their intracellular trafficking, which is currently under review by Adv Drug Deliv Rev Journal.
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| 今後の研究の推進方策 |
The in vivo evaluation and optimization of the promising delivery systems will be performed in a real model of liver fibrosis demonstrating high activation of HSCs. The animal model will be subjected to in-depth histo-pathological, biochemical, and genetic evaluation to confirm that it can reflect the real microenvironment of the human liver fibrosis. The systemic intravenous administration will be adopted in all experiments for a real representation of the clinical situation. The lipid composition and physico-chemical properties of the developed lipid nanoparticles will be extensively manipulated to control their in vivo performance. The top-performing candidate will be subjected to a second round of in vivo evaluation to judge its mRNA functional delivery efficiency to the various organs, robustness, intra-hepatic selectivity, and potency. Moreover, the in vivo biosafety will be evaluated histo-pathologically and biochemically at clinically-relevant high mRNA doses. Eventually, a mechanistic investigation of the gene delivery to HSCs, cellular uptake, intracellular kinetics, and protein production is planned. The promising in vivo results will be considered if a patent application will be submitted. The results are planned to be published in a high impact factor journal and to be announced in various scientific events.
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