Engineering desialylation- and CD42 cleavage-resistant platelets to optimise storage and maintain lifespan and function following transfusion.
A K Waller(1) A Crosby(1) W WY Lau(1) K Mahbubani(1) K Saeb-Parsy(1) R Li(2) C Ghevaert(1)
1:University of Cambridge; 2:Emory University
Transfusion of platelets to either prevent or treat bleeding and maintain haemostasis, has been an effective therapy especially in patients undergoing chemotherapy or major surgery. Whilst an established therapy, platelet transfusion does present several difficulties, for example potential bacterial contamination due to the need to store platelet units at room temperature to prevent cold storage-induced lesions that reduce function and survival in circulation. Here, we propose to use CRISPR Cas9 editing of induced pluripotent stem cells (iPSCs), to (a) knock-out the metalloproteinase ADAM17 and two sialidases present in megakaryocytes (MKs) and (b) alter residues in CD42 where ADAM17 cleaves it. Megakaryocytes will be produced from iPSCs by a forward programming method that relies on the expression of key transcription factors under the control of an inducible cassette. The genome editing of the trigger region and mechanosensory domain of GP1BA will be performed to remove the cleavage site for ADAM17 whilst retaining the resting configuration of the mechanosensory domain and its ability to unfold when bound to vWf at high shear. We hypothesise that the platelet progeny from the engineered iPSCs would allow them to be stored at lower temperatures and for longer periods, thus reducing wastage and the risk of bacterial contamination. Additionally, by knocking out the two sialidases (neu1 and neu3) we will produce platelets that maintain surface sialylation in circulation thereby prolonging their survival and haemostatic function. Finally we will perfuse labelled platelets produced in vitro in an ex vivo human spleen model to measure their survival.