Report on the Compatibility of mRNA-Lipid Nanoparticle-Based Gene-Engineered Platelets with Blood Banking Practices

Academic Background

Platelets play an essential role in hemostasis, inflammation, sepsis, and cancer. However, clinical applications of platelet transfusions are primarily limited to managing thrombocytopenia and bleeding. To broaden the scope of platelet transfusions, researchers aim to genetically engineer platelets with new or enhanced functionalities. Previous studies have demonstrated that lipid nanoparticles containing mRNA (mRNA-LNPs) can genetically modify platelets in non-clinical crystalloid solutions. However, platelets intended for transfusion are typically stored in plasma or plasma supplemented with platelet additive solutions (PAS) at either room temperature or 4°C (for acute bleeding scenarios). Therefore, developing an mRNA-LNP system capable of directly transfecting platelets in plasma or PAS—while maintaining compatibility with current blood banking practices—is of significant clinical importance.

Paper Source

This study, conducted by a multidisciplinary team of researchers—Colton Strong, Jerry Leung, Emma Kang, among others—was carried out in institutions like the University of British Columbia, Versiti Blood Research Institute, and Nanovation Therapeutics. The paper was published on November 21, 2024, in the journal Blood.

Research Process and Findings

Research Workflow

1. Platelet Collection and Storage
   Two types of platelet concentrates were used: one pooled from four ABO-matched donors, suspended in 100% plasma, and a second pooled from seven ABO-matched donors, suspended in 70:30 (PAS:plasma) by volume. Platelets were transfected on the first day after collection.

2. Preparation and Characterization of mRNA-LNPs
   The researchers systematically optimized the mRNA-LNP formulation, screening various ionizable lipids, structural phospholipids, and PEGylated lipids to determine the most effective formulation for transfecting platelets in plasma and PAS. Platelet function and morphology were characterized using flow cytometry, rotational thromboelastometry (ROTEM), and transmission electron microscopy.

3. Platelet Transfection and Functional Testing
   Platelets were transfected with mRNA-LNP in 100% plasma, PAS70:30, and 100% PAS solutions. The study assessed the impact of lipid composition on transfection efficiency and evaluated platelet activation, LNP uptake, and functionality via flow cytometry.

4. Platelet Storage Experiments
   Transfected platelets were stored at room temperature (RT) with agitation or at 4°C without agitation. Over various time points, baseline activation, mRNA expression, platelet count, gas analysis, and clotting parameters were measured to evaluate the effects of storage on mRNA-LNP-transfected platelets.

Key Findings

1. Transfection in Clinical Storage Solutions
   The optimized mRNA-LNP formulation, composed of the ionizable lipid NTX-001, structural phospholipid POPC, and 0.5% DMG-PEG2000, achieved high transfection efficiency in plasma and PAS70:30. Compared to prior formulations, transfection efficiency improved 3.3-fold in PAS70:30 and 4.1-fold in plasma.

2. Platelet Functional Stability and Storage
   Transfected platelets retained similar coagulation ability and morphology compared to non-transfected controls. During storage, platelets maintained mRNA expression and functionality without significant differences between transfected and non-transfected platelets. At room temperature, mRNA expression increased over the first week, while platelets at 4°C showed peak expression on day one, which declined gradually but remained detectable up to 14 days.

3. Scalability of Transfection
   mRNA-LNP transfection technology was scalable to both physiological (250 × 10^6/mL) and supraphysiological (800 × 10^6/mL) platelet concentrations. Transfected platelets maintained normal activation and aggregation profiles, even at the higher platelet concentrations used in transfusion settings.

Conclusions and Implications

This study successfully developed an mRNA-LNP system capable of directly transfecting platelets in plasma and PAS storage solutions, compatible with current blood banking practices. This technology has promising potential to expand clinical platelet therapies, such as engineering platelets to express antifibrinolytic factors or anti-cancer agents. Additionally, the study lays the groundwork for future development of mRNA-LNP-based platelet products and cell therapies.

Research Highlights

1. Innovative Approach: The study is the first to facilitate direct transfection of platelets in clinical-grade plasma and PAS solutions, overcoming the limitations of non-clinical crystalloid storage systems.
2. Clinical Potential: The compatibility of the optimized mRNA-LNP system with blood storage practices allows practical implementation in healthcare settings.
3. Scalability and Stability: The study demonstrates successful transfection of platelets at physiological and transfusion-relevant concentrations, with stable functionality during storage.

Additional Insights

The study investigated how different lipid components affected transfection efficiency. Notably, the saturation state of structural phospholipids and the molar ratio of PEGylated lipids significantly influenced transfection efficacy. Plasma proteins like albumin were found to stabilize platelets and reduce activation during the transfection process.

This research represents a significant step forward in platelet gene engineering and cell therapy, offering broad scientific and clinical applications.