PB0359 - CRISPR genome editing coupled with optimized biomanufactured human platelets for molecular and functional investigation of platelet biogenesis.

Background: Many rodent models represent powerful tools for our understanding of platelet disorders. However, the inherent differences between human and murine platelets limit their exploration. These drawbacks, combined with the difficulty in accessing patients and the desire to reduce animal experimentation, make CRISPR genome editing in CD34+ haematopoietic progenitors (HP) an innovative and powerful tool to better explore platelet pathologies and for future therapeutic applications.

Aims: Our goal was to develop an efficient method for large-scale production of genetically modified platelets, proceeding from CRIPSR-modified HP down to platelet production. As a proof of concept, we deleted the genes encoding GP1BA and TUBB1, known to play essential roles in platelet production and function.

Methods: Blood-derived CD34+ HP were inactivated for GP1BA or TUBB1 using CRISPR/Cas9. MKs were then differentiated and subjected to a custom proprietary platelet release device (see accompanying abstract).

Results: CRISPR editing resulted in a 80-90% deletion efficiency for each targeted gene. This resulted in MK maturation defects previously identified in patients and mutant mice. These included for GP1BA, a less well-developed demarcation membrane system and abnormal proplatelet morphology, characterised by fewer pseudopodial extensions, thicker shaft sections and increased diameter of the coiled elements. As expected for TUBB1, a profound defect in proplatelet extension was observed. For both deletions, fully mature MK when subjected to the platelet release device led to biomanufactured platelets exhibiting larger diameters and misarranged microtubule coils. Large production of these modified platelets is under way which will allow their functional evaluation.

Conclusion(s): These results predict that the CRIPSR/Cas9 technology combined with our platelet release device will represent a powerful tool for studying candidate genes involved in human platelet biogenesis and function.