Background: Platelets are transfused therapeutically for hemostasis, and are an integral part of hemorrhage management. However, transfusions can be ineffective in the most severe cases of hemorrhage. In addition to hemostasis, platelets are a potential cell therapy in other applications, but development has been hindered by inadequate methods to control which proteins are expressed by platelets. For example, there are no methods to express exogenous proteins in transfusable platelets, which would expand their use to help treat the diseases they modulate. A method is therefore needed to modify transfusable platelets, and thus enhance their protein composition for specific applications.

Aims: To produce engineered, transfusable platelets to enhance their natural coagulability and functional repertoire by directly transfecting donor-derived platelets and megakaryocytes with mRNA via lipid nanoparticle (LNP)-mediated delivery. LNPs have already demonstrated clinical safety and efficacy for gene therapy, and cultured megakaryocytes provide an alternative source of cells that can be engineered to produce modified platelets.

Methods: As a proof-of-concept, megakaryocytes cultured from cord-blood hematopoietic stem cells were treated with GFP-encoding mRNA-LNPs and induced to produce platelet-like particles, with nonGFP mRNA-LNPs as a control. Donor-derived platelets were also incubated with GFP or luciferase-encoding mRNA-LNPs, or LNPs incorporating the lipophilic tracer DiI. GFP expression and LNP uptake were assessed following 24 hours via flow cytometry and confocal microscopy; luciferase expression was assessed by measuring luminescence intensity normalized to total protein content.

Results:

(A) Percentage-positive platelet-like particles expressing GFP and (B) corresponding change in median fluorescence intensity.Figure 1.

Donor-derived platelet mRNA-LNP expression of (A) luciferase and (B) GFP after 24 hours. Error bars represent SEM.Figure 2.

 

 

Megakaryocytes demonstrate successful transfection and produce GFP-positive plalelet-like particles. Nearly 100% of donor-derived platelets appear to take up the LNPs, and demonstrate expression of both GFP and luciferase.

Conclusions: Megakaryocytes and platelets can readily be transfected with LNPs to produce exogenous protein. Further optimization can eventually lead to the creation of a platform technology that in the long-term will allow platelets to deliver therapeutic proteins and yield more effective platelet products.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/enhancing-transfusable-platelets-using-mrna-therapy-to-produce-exogenous-proteins-2/