PB0726 - Genetic engineering of megakaryocytes from blood progenitor cells using mRNA lipid nanoparticles

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Jerry Leung, PhD

Postdoctoral Research Fellow
The University of British Columbia
Vancouver, British Columbia, Canada

Co-Author(s)

AP

Asel Primbetova

Postdoctoral Research Fellow
The University of British Columbia
Vancouver, British Columbia, Canada
CS

Colton Strong

PhD Candidate
The University of British Columbia
Vancouver, British Columbia, Canada
AH

Andrew Hagner

Postdoctoral Research Fellow
The University of British Columbia
Vancouver, British Columbia, Canada
DD

Dana Devine

Professor
The University of British Columbia
Vancouver, British Columbia, Canada
PC

Pieter R. Cullis

Professor
The University of British Columbia
Vancouver, British Columbia, Canada
PZ

Peter W. Zandstra

Professor
The University of British Columbia
Vancouver, British Columbia, Canada
Christian J. Kastrup

Senior Investigator
Versiti Blood Research Institute
Milwaukee, Wisconsin, United States

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 harbouring 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 non-viral techniques. Lipid nanoparticles (LNP) are the gold standard in non-viral delivery systems, as demonstrated in their clinical application in SARS-CoV-2 vaccines, and could represent a facile strategy to modify MKs with a variety of nucleic acid payloads.

Aims: To investigate whether (1) LNP resembling the clinically approved LNP formulations can transfect cultured hematopoietic stem/progenitor cell (HSPC)-derived MKs to express exogenous proteins and (2) elicit functional changes when expressing a representative physiologically relevant protein.

Methods: Cord-blood derived HSPCs were differentiated into MKs and treated with lipid nanoparticle formulations containing mRNA (mRNA-LNP). Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein (EGFP). Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor VII (FVII).

Results: LNP enabled transfection efficiencies of 95% in MKs (Fig. 1A) and did not impair MK maturation. MKs engineered to express exogenous FVII yielded a decreased clot time and increased α-angle in FVII-deficient plasma following clot initiation (Fig. 1B,C).

Conclusion(s): HSPC-derived MKs can be engineering with high transfection efficiency using lipid nanoparticles to express exogenous proteins and elicit functional changes. This approach provides an easy-to-use modular platform to genetically modify MKs, which can be potentially extended to producing genetically modified cultured platelets.