Leung Jerry, Primbetova Asel, Strong Colton, Hay Brenna N, Hsu Han Hsuan, Hagner Andrew, Foster Leonard J, Devine Dana, Cullis Pieter R, Zandstra Peter W, Kastrup Christian J
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada; Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada; NanoMedicines Research Group, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia, Canada.
J Thromb Haemost. 2025 Jan;23(1):306-313. doi: 10.1016/j.jtha.2024.09.008. Epub 2024 Sep 26.
Platelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harboring lineage-driven transgenes and inducing the production of in vitro modified platelets. The use of viruses, however, poses challenges in clinical implementation, and no methods currently exist to genetically modify MKs with nonviral techniques. Lipid nanoparticles (LNPs) are a nonviral delivery system that could enable a facile strategy to modify MKs with a variety of nucleic acid payloads.
To investigate whether LNPs can transfect cultured hematopoietic stem/progenitor cell-derived MKs to express exogenous proteins and induce functional changes.
MK and MK progenitors differentiated from cord blood-derived hematopoietic stem/progenitor cells were treated with LNP formulations containing messenger RNA and resembling the clinically approved LNP formulations. Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein. Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor (F)VII, a representative physiologically relevant protein.
LNPs enabled transfection efficiencies of 99% in MKs and did not impair MK maturation, viability, and morphology. MKs engineered to express exogenous FVII decreased clotting time in FVII-deficient plasma following clot initiation.
This approach provides an easy-to-use modular platform to genetically modify MK and MK progenitors, which can be potentially extended to producing genetically modified cultured platelets.
血小板是出血控制和管理的重要组成部分，对血小板进行工程改造以表达治疗性蛋白质可扩大其作为细胞疗法的应用。通过修饰血小板前体细胞巨核细胞（MKs）可实现基因工程化血小板。当前策略包括在体外使用携带谱系驱动转基因的病毒载体转染MK祖细胞，并诱导产生体外修饰的血小板。然而，病毒的使用在临床应用中带来了挑战，目前尚无利用非病毒技术对MKs进行基因修饰的方法。脂质纳米颗粒（LNPs）是一种非病毒递送系统，可提供一种简便策略，用多种核酸载荷修饰MKs。
研究LNPs能否转染培养的造血干细胞/祖细胞来源的MKs以表达外源蛋白并诱导功能变化。
用含有信使核糖核酸且类似于临床批准的LNP制剂的LNP制剂处理从脐带血来源的造血干细胞/祖细胞分化而来的MK和MK祖细胞。通过流式细胞术检测增强型绿色荧光蛋白的表达来评估转染效率。通过旋转血栓弹力图检测外源凝血因子（F）VII（一种具有代表性的生理相关蛋白）的表达来评估MKs的功能变化。
LNPs使MKs的转染效率达到99%，且不损害MKs成熟、活力及形态。经工程改造表达外源FVII的MKs在凝血启动后可缩短FVII缺陷血浆中的凝血时间。
该方法提供了一个易于使用的模块化平台，用于对MK和MK祖细胞进行基因修饰，这有可能扩展到生产基因修饰的培养血小板。
