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. 2025 Jan;23(1):306-313.
doi: 10.1016/j.jtha.2024.09.008. Epub 2024 Sep 26.

Genetic engineering of megakaryocytes from blood progenitor cells using messenger RNA lipid nanoparticles

Jerry Leung  1 , Asel Primbetova  2 , Colton Strong  3 , Brenna N Hay  4 , Han Hsuan Hsu  2 , Andrew Hagner  2 , Leonard J Foster  3 , Dana Devine  5 , Pieter R Cullis  6 , Peter W Zandstra  7 , Christian J Kastrup  8
Affiliations

Genetic engineering of megakaryocytes from blood progenitor cells using messenger RNA lipid nanoparticles

Jerry Leung et al. J Thromb Haemost. 2025 Jan.

Abstract

Background: 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.

Objectives: To investigate whether LNPs can transfect cultured hematopoietic stem/progenitor cell-derived MKs to express exogenous proteins and induce functional changes.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: cell therapy; genetic engineering; mRNA; megakaryocytes; nanoparticle drug delivery system.

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Conflict of interest statement

Declaration of competing interests P.R.C. and C.J.K. are cofounders of and hold equity in NanoVation Therapeutics Inc. P.R.C. and C.J.K. are cofounders of and hold equity in SeraGene Therapeutics Inc. C.J.K. is a cofounder of and holds equity in CoMotion Drug Delivery Systems Inc. P.R.C. is a cofounder of and holds equity in Acuitas Therapeutics. P.W.Z. is a cofounder of and holds equity in Notch Therapeutics. P.W.Z. is the Canada Research Chair in Stem Cell Engineering. J.L., C.S., P.R.C., and C.J.K. have submitted intellectual property on RNA-LNP. A.P., B.N.H., H.H.H., A.H., L.J.F., and D.D. have no conflicts of interest to declare.

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