PL5-SN-27 - Genetic Engineering of Megakaryocytes From Blood Progenitor Cells Using mRNA Lipid Nanoparticles

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. The aims of the present study are to thus 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.


Study

Design/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). Data was analyzed by one- or two-way ANOVA corrected with a post-hoc Tukey or Bonferroni test. P-values < 0.05, 95% confidence intervals, were considered significant.


Results/Findings:

MKs treated with any of the three mRNA-LNP encoding for EGFP all expressed EGFP (Fig. Ai, ii), with up to 99% of MKs positive for GFP. MKs treated with SM-102 mRNA-LNP yielded the highest EGFP expression, with approximately 15-fold higher expression than MC3, which yielded the lowest (1346670 ± 58405 A.U. versus 91822 ± 13995 A.U. respectively, P< 0.01). MKs engineered to express exogenous FVII (FVII-MK) decreased the clot time by one-third (184 ± 18 s) compared to untreated MKs (UT-MK, 286 ± 5 s, P< 0.01) or MKs treated with EGFP-LNP (EGFP-MK, 285 ± 5 s, P< 0.01) in FVII-deficient plasma following clot initiation (Fig. Aiii). The α-angle was similarly significantly increased with FVII-expressing MKs compared to untreated or EGFP-expressing MKs (76º ± 0.9º versus 68º ± 0.8º and 68º ± 0.3º) (Fig. Aiv).

Conclusions:

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.