Epigenome editing of human hematopoietic stem cells: reduced platelet aggregation after differentiation and sustained gene suppression
Gene regulation at the starting point of platelet production
A preclinical study found that suppressing gene expression in human hematopoietic stem and progenitor cells (HSPCs) with epigenome editing also reduced the aggregation capacity of platelets derived from those cells. The team used the editing tool CHARM to suppress ITGB3 expression and examined whether suppression persisted through cell proliferation and differentiation and after transplantation into mice. The study is a bioRxiv preprint; it is not a clinical study testing whether the approach prevents thrombosis in people.
Antiplatelet drugs such as aspirin and clopidogrel are used to prevent and treat thrombosis. However, they must be taken daily over the long term, and difficulties with adherence and drug resistance in some patients can limit their effectiveness. The team focused on the fact that platelets are continually produced from hematopoietic stem cells (HSCs) through megakaryocytes. They therefore tested whether gene suppression established in the starting cells would persist in their downstream progeny.
ITGB3 suppression and persistence 16 weeks after transplantation
The team delivered messenger RNA (mRNA) encoding CHARM and guide RNA specifying the target into human CD34+ HSPCs by electroporation. Rather than changing the DNA sequence, CHARM induces methylation at the target site to suppress gene expression. ITGB3 encodes a component of integrin αIIbβ3, a receptor involved in platelet aggregation.
When the edited cells were differentiated into mature megakaryocytes, an average of about 90% were negative for CD61, the ITGB3 protein. This figure does not represent a thrombosis-prevention rate or a reduction in aggregation. In the functional assay, platelets derived in culture were mixed with platelets from healthy donors and stimulated with an aggregation-inducing agent. Platelets derived from ITGB3-suppressed cells showed less capacity to aggregate with donor platelets than did controls.
Target-site methylation persisted during long-term culture and through three rounds of serial colony replating. In an experiment transplanting human HSPCs into immunodeficient mice, methylation was maintained in human CD34+ cells recovered 16 weeks later. After these cells were differentiated into megakaryocytes again, an average of about 80% were CD61-negative. Engraftment and the composition of major blood-cell lineages were also similar to those in controls. What was demonstrated here was suppression of expression in recovered and redifferentiated cells, rather than a direct measurement of circulating platelet function in the mice.
A separate tool was used to reverse gene suppression. When mRNA for the demethylation tool TET1-dCas9 was delivered to CHARM-treated cells by a second electroporation, target-site methylation decreased and CD61 expression recovered. This was not an experiment in which CHARM itself was used both to establish and to reverse suppression.
Thrombosis prevention and bleeding safety remain to be tested
The study shows that gene suppression induced by a transiently delivered editing tool can persist after cell proliferation and differentiation. The team also explored the approach with other platelet-related genes, including GP1BB, ANO6, and SERPINH1, observing suppressed expression and functional changes related to each target.
However, human platelet production was low in the transplanted mice, so the researchers could not assess platelet function or thrombus formation in vivo. The study also did not establish whether the effect lasts for life or how to reverse suppression in a patient's body. Because loss of ITGB3 function is associated with bleeding disorders, the effect of inhibiting aggregation on hemostasis also needs to be tested.
Molecular analyses showed increased expression of the neighboring gene MYL4. DNA methylation-based suppression also requires regulatory regions that are rich in CpG sites and have low baseline methylation. Further development will need to examine target-specific bleeding risks and unintended changes in gene expression, alongside persistence.
The results and experimental conditions are available in the full paper.
Thrombosis remains a major cause of cardiovascular and cerebrovascular diseases, driven in large part by platelet activation and aggregation. Because platelets are continuously produced from hematopoietic stem cells (HSCs), durable reprogramming of HSC output offers a unique opportunity for a one-time antithrombotic intervention. Here, we show that DNA methylation-based epigenome editors delivered transiently as RNA result in stable, heritable gene silencing in primary human HSCs that persists through long-term self-renewal and megakaryocytic differentiation, while remaining reversible through targeted demethylation. Targeting the platelet integrin β3 (
This study is a preclinical proof of concept linking gene suppression established in hematopoietic stem and progenitor cells to functional changes in differentiated platelets. In future cell-therapy development, it could inform studies comparing the strength and persistence of suppression across ITGB3 and other platelet targets, and finding a balance between inhibiting aggregation and preserving hemostasis. The recovery of expression using a separate demethylation tool also provides a starting point for developing ways to reverse suppression. Before patient use, further work must test reductions in thrombosis in vivo, bleeding safety, delivery and transplantation of edited cells, and methods for restoring expression. The current results do not support replacing existing antiplatelet drugs or concluding that a single treatment could provide long-term prevention.