CO005 | HEPATIC ENDOTHELIUM AS A SAFE AND DURABLE TARGET FOR LENTIVIRAL GENE THERAPY OF HEMOPHILIA A

E. Borroni, F. Antony, S. Merlin, V. Kalandadze, C. Borsotti, A. Follenzi | Università del Piemonte Orientale, Novara, Italy

Background and Aims. Hemophilia A (HA) is a bleeding disorder caused by mutations in the F8 gene, leading to reduced/absent activity of factor VIII (FVIII). Current FVIII replacement therapies are limited by inhibitor development and the need for lifelong treatment. Gene therapy has recently reshaped the therapeutic landscape, with EMA and FDA approval of AAV-based hepatocyte-directed approaches for severe HA. However, clinical data show a progressive decline in FVIII expression over time. In addition, recent preclinical studies in mice suggest that supraphysiological FVIII production in hepatocytes may activate the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, contributing to this loss of efficacy. Since liver sinusoidal endothelial cells (LSECs) are the main physiological source of FVIII, they represent an attractive target for gene correction. In our preclinical studies, FVIII expression in LSECs using a lentiviral vector (LV) under the control of the LSEC-specific Stabilin2 promoter resulted in stable correction of the bleeding phenotype in a mouse model, with sustained FVIII activity and no inhibitor formation. The aim of this study was to assess whether LV-directed FVIII expression in LSECs could affect liver homeostasis and tissue integrity over time when gene therapy is applied to hemophilic newborns and adult mice.
Methods. Neonatal and adult HA mice were injected with lentiviral vector (LV) encoding FVIII under the LSEC-specific Stabilin2 (STAB2) promoter. Phenotypic correction has been assessed by aPTT and anti-FVIII ELISA assays, while liver integrity was evaluated by histology and immunofluorescence before and after an additional endothelial damage induced with monocrotaline (MCT, 150 mg/kg). To determine whether FVIII expression in LSECs activates endoplasmic reticulum (ER) stress pathways, unfolded protein response (UPR) markers (BIP, XBP1s, ATF4, CHOP) were quantified by qRT PCR and western blot at short- and long-term timepoints after LV administration.
Results. In both neonatal and adult HA mice, LV.STAB2 FVIII achieved stable FVIII activity and maintained constant expression even after MCT-induced endothelial damage, with vector integration profiles remaining comparable between the LV and LV+MCT groups. To assess whether FVIII expression in LSECs affects liver homeostasis, we evaluated ER stress and tissue integrity. Adult HA mice injected with LV. STAB2-FVIII showed constant FVIII activity throughout follow-up without inhibitors. Untreated HA mice exhibited elevated hepatic UPR markers, suggesting baseline ER stress associated with the disease. In contrast, LSEC-specific FVIII expression significantly reduced UPR marker expression, restoring levels closer to WT. Histological and immunofluorescence analyses showed preserved liver architecture, absence of inflammatory infiltrates, and no evidence of endothelial or parenchymal injury, supporting the safety of LSEC-directed gene therapy.
Conclusions. LSEC-targeted FVIII gene therapy provides efficient and sustained correction of the hemophilic phenotype without inducing cellular stress or compromising liver integrity. These findings support the feasibility of LSEC-targeted FVIII gene therapy as a safe and durable strategy to maintain long-term FVIII activity while preserving liver homeostasis