CO085 | GENE REGULATION OF THE HYPERACTIVE FIX PADUA VARIANT VIA DCAS9-KRAB TO MODULATE THROMBOPHILIA

A. Benetti¹, C.M. Radu¹, M. Trevisan², C. Samà¹, C. Simion¹, E. Campello¹, P. Simioni¹ | ¹First Chair of Internal Medicine and Thrombotic and Haemorrhagic Diseases Unit, Department of Medicine, Padova University Hospital, Padova, Italy; ²Department of Molecular Medicine, University of Padua, Italy

Background and Aims. Thrombophilia is characterized by an increased risk of venous thromboembolism due to a hypercoagulable state resulting from inherited or acquired alterations in coagulation or fibrinolysis. Among genetic variants, factor IX (FIX) R384L (FIX Padua) is associated with markedly increased FIX activity and a consequent elevated thrombotic risk. To modulate FIX expression and activity in carriers of the FIX Padua variant, CRISPR/dCas9-KRAB–mediated transcriptional repression represents a reversible and dynamic epigenetic strategy with the potential to decrease thrombotic risk. This study aimed to evaluate CRISPR/dCas9-KRAB-mediated modulation of the Factor IX Padua variant expression in an “ex vivo” model of human hepatocyte-like cells (HLCs) derived from a variant carrier.
Methods. Peripheral blood stem cells were isolated from a thrombophilia patient carrying the FIX Padua variant and reprogrammed into induced pluripotent stem cells (iPSCs). Clonal iPSC lines were established, and pluripotency was confirmed by the expression of Oct4, Sox2, Klf4, and Nanog. Gene modulation was performed using CRISPR/dCas9-KRAB technology. iPSCs were subsequently differentiated into HLCs, using a previously optimized protocol. FIX expression was assessed by immunofluorescence analysis (IF).
Results. iPSCs exhibited alkaline phosphatase activity and expressed pluripotency markers, as confirmed by IF. Upon differentiation, HLCs expressed cytokeratin 18, as demonstrated by IF and real-time qPCR. FIX expression in patient-derived HLCs was assessed before and after CRISPR/dCas9-KRAB–mediated repression. Prior to editing, HLCs showed high FIX expression levels, which were significantly reduced by approximately 40% following CRISPR/dCas9-KRAB modulation, as quantified by mean fluorescence intensity.
Conclusions. This study demonstrates that CRISPR/dCas9-KRAB–mediated transcriptional repression effectively modulates the expression of a hyperactive coagulation factor in an “ex vivo” model derived from a carrier of the FIX Padua. This reversible epigenetic approach enables precise regulation of FIX levels without introducing permanent genomic alterations. The observed reduction in FIX expression in edited HLCs supports the potential of this strategy to mitigate prothrombotic risk. Overall, these findings highlight CRISPR/dCas9-KRAB–based gene regulation as a promising approach for personalized therapies for inherited thrombophilia.