Abstract
Introduction: Haemophilia A (HA) is a rare X-linked recessive bleeding disorder caused by variants in the F8 gene, which encodes coagulation factor VIII (FVIII). The severity of HA is determined by the level of plasma FVIII activity and is classified as severe (<1%), moderate (1%–5%), or mild (5%–40%). In patients with HA, increasing FVIII activity to above 2–5% can significantly improve quality of life.
The aim of this study was to restore FVIII levels to prevent spontaneous bleeding by correcting FVIII variants using CRISPR-Cas9 base editing in liver sinusoidal endothelial cells (LSECs), which are recognized as a key FVIII producers’ cells.
Methods: Peripheral blood mononuclear cells (PBMC) from 3 HA patient and 1 healthy subject were isolated and reprogrammed into induced pluripotent stem cells (iPSC) after transduction in a feeder-free culture with a non-integrative system with Sendai virus. The clones obtained were stabilized using an enzymatic method with EDTA and characterised for their pluripotency markers expression. Gene correction of the HA-iPSCs will be carried out using CRISPR-Cas9 DNA base editing technologies with specially target of FVIII variations. The following gene variants NM_000132.4:c.6593G>T, NM_000132.4:c.4285C>T and NM_000132.4:c.5373+1G>A were considered. HA-iPSCs will be differentiated into LSECs using an optimized differentiation protocol involving BMP4 and VEGF and others growth factors. Specific cells markers were analysed using immunofluorescence (IF) and real-time PCR (RT-qPCR).
Results: Obtained iPSCs was positive for alkaline phosphatase staining and immunofluorescence showed the expression of stem cells markers (Oct4, Sox2, Klf4 and Nanog). The LSECs differentiated obtained cells were stained positive for the expression of CD31, STAB2 and FCGR2B. Following RT-qPCR and flow cytometry analysis expression of the above markers confirmed the obtained cells phenotype. Furthermore, the expression of FVIII were assessed by IF and flow cytometry in the HA-patients obtained cells before and after gene editing and the correction was confirmed.
Conclusions: In this study, we generated specific iPSCs from HA-patients carrying different F8 gene variants, derived from PBMCs. These iPSCs were then differentiated in vitro into LSECs using optimized protocols. CRISPR-Cas9 mediated gene correction enabled us to demonstrate that specific disease-causing variants can be effectively repaired, resulting in the restoration of FVIII expression.
The LSECs obtained through these protocols represent a useful in vitro model for evaluating FVIII restoration in HA-patients with different F8 gene variants. Given the limitations of current treatments, new strategies for a definitive cure are urgently needed. Gene and cell therapies offer promising avenues and could provide a powerful, personalized therapeutic approach to optimize treatment for each individual HA-patient.