Title Transplantation of fetal liver cells into newborn hemophilic mice for the treatment of hemophilia A without inhibitors formation Simone Merlin, Silvia Buzzi, Tamara Garcia-Leal, Maria Jose Sanchez, Antonia Follenzi Background. Hemophilia A cell therapy approaches in paediatric individuals require suitable FVIII-producing cell populations presenting stable engraftment potential. We previously showed that adult-derived FVIII-producing cells, i.e. liver sinusoidal endothelial cells (LSEC) and hematopoietic stem cells (HSC), can be used for the treatment of adult hemophilia A (HA) mice. However, after transplantation in busulfan-conditioned newborn mice, adult LSEC/HSC cannot efficiently engraft, while murine fetal liver (FL) hemato/vascular cells from day 11-13 of gestation (E11-E13) strongly reconstitute the hematopoietic compartment and showed multiorgan endothelial reconstitution potential while secreting FVIII. Aims. To investigate the engraftment of FL cells in newborn HA mice as a new strategy to develop an experimental treatment for HA in neonates. Materials and Methods. We transplanted E11-E13 GFP+ FL cells using intravenous cell transfer into myelo-ablated newborn syngeneic HA mice. The engraftment level as well as the FVIII production and activity was assessed in HA recipients starting from 4 weeks and followed-up to 1 year after transplantation. Moreover, we evaluated the presence of anti-FVIII antibodies/inhibitors in plasma of transplanted mice. Results. We transplanted FL cells from E11-E13 GFP+ mice into newborn HA mice pre-treated with busulfan (FLE11-E13+BU). Control groups received FL E11-E13 GFP+ cells without busulfan pretreatment (FLE11-E13noBU) or PBS ± busulfan. In FLE11-E13+BU group we observed >60% chimerism in peripheral blood with a concomitant FVIII activity (up to 16%) which remained stable up to 1 year after transplantation. In FLE11-E13noBU we observed low level of engraftment (¿10%) and a consequent low FVIII activity (¿3.5%). Both groups of mice, FLE11-E13+BU and E13noBU, did not develop anti-FVIII antibodies, not even after FVIII immunization. Conclusions. Transplantation of FL cells may provide a novel and highly promising neonatal preclinical models, for HA treatment, paving the way for studies aiming at deriving long-term reconstituting ¿fetal-like¿ hemato/vascular progenitors from other sources (e.g. iPS)

Resumen del trabajo presentado en el 10th BIC International Conference, celebrado en Genoa (Italia), del 6 al 8 de septiembre de 2019