Abstract Number: PB1724
Meeting: ISTH 2020 Congress
Background: Platelets are ideal therapeutic carriers given systemic circulation and concentration at sites of injury. Platelet microparticle (PMP) generation and transfer of endogenous miRNA to nucleated cells via PMPs are well documented. However, investigators have had limited success introducing exogenous, potentially therapeutic miRNA to platelet carriers using common methods.
Aims: To assess rapid, high efficiency cationic lipid transfection of the tumor suppressor miR-34a and an siRNA control into platelets; and to assess the impact of transfection on platelet activation and function; and to define methods for preserving transfected platelets.
Methods: Platelet transfection with miR-34a and an siRNA control was performed using a cationic lipid formulation and proprietary transfection medium over 30 minutes. Platelets were cryopreserved by rapid freezing and maintained at -80oC until thawed. Lyopreserved platelets were frozen and dried under vacuum, and stored at room temperature until reconstituted. Fresh and preserved transfected platelets were evaluated by cell count, flow cytometry, light transmission aggregometry, fluorescence microscopy, and occlusion of a thromboplastin-collagen coated microcapillary under shear.
Results: There were no observed differences on platelet physiology with transfection. Both control and transfected platelets maintained low P-selectin expression (Table 1). Nearly all platelet events were transfected and showed intracellular accumulation of labeled RNA. Transfected platelets responded normally to common platelet agonists. Cryopreserved platelets exhibited quantitative cell recovery and high retention of transfected cargo. Lyopreserved platelets maintained high cell recovery (75%) but poor retention of transfected material. All transfected platelet products occluded a coated microcapillary within 15 minutes.
Conclusions: Platelets may be transfected with high efficiency and low toxicity using a tumor suppressing short RNA. Transfected platelets preserved by cryopreservation retain a substantial portion of transfected RNA. All transfected platelet products retain hemostatic function. Our results provide support for the investigation of platelets and preserved platelet products as vehicles for the delivery of short RNA therapeutics.
|Parameter||Transfected Platelets||Control Platelets|
|Platelet P-selectin Expression (%)||5.51||4.14|
|Aggregation with Collagen 10 µg/mL (%)||66.0||88.9|
|Aggregation with Arachidonic Acid 50 µg/mL (%)||68.5||89.2|
|Aggregation with Thrombin 2.5 U/mL (%)||83.0||92.0|
|Cryopreserved Platelet siRNA Retention (%)||90||n/a|
|Lyophilized Platelet siRNA Retention (%)||15||n/a|
|Capillary Occlusion Time: Fresh PRP (mm:ss)||15:16||10:07|
|Capillary Occlusion Time: Cryopreserved Platelets (mm:ss)||09:32||10:55|
|Capillary Occlusion Time: Lyophilized Platelets (mm:ss)||14:52||12:54|
[Table 1: Evaluation of Transfected and Control Platelets]
To cite this abstract in AMA style:Lee A, Sheik D, Moskowitz KA. High Efficiency Transfection and Preservation of Platelets with Tumor Suppressing Short RNA [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/high-efficiency-transfection-and-preservation-of-platelets-with-tumor-suppressing-short-rna/. Accessed January 27, 2022.
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