Abstract
<title>Abstract</title> <p>In vivo liver-directed gene therapy (GT) is a therapeutic option for the permanent correction of monogenic diseases. Lentiviral vectors (LV), which integrate into the target cells' genome, represent a unique platform for intervention in childhood, with negligible limitations from pre-existing immunity to the parental virus. However, adaptive immunity to the encoded transgene and vector components may hamper the efficacy of the therapy and its potential re-administration. In a mouse model of hemophilia A, an inherited disease due to lack of coagulation factor VIII (FVIII) activity, we tested variants of the FVIII transgene with increased expression (codon-optimized) and half-life (XTENylated) to reconstitute in vivo therapeutic FVIII amounts with a minimally effective LV dose, revealing a direct correlation between FVIII productivity per integrated vector copy and immunogenicity. Maximizing the proportion of LV-engineered hepatocytes with low output per LV copy established immune tolerance to FVIII, thereby stably maintaining therapeutically relevant levels. Conversely, using LV with high FVIII output required co-administration of an immunomodulatory regimen to transiently block T cell costimulatory pathways and achieve stable reconstitution of FVIII and immune tolerance. Moreover, this regimen prevented the development of vector-neutralizing immune responses, enabling GT redosing and adjustment of the transgene output based on the outcome of the first dose. This strategy could also be extended to adeno-associated-vector-mediated liver gene transfer. Overall, this study reveals key determinants of GT-evoked immunity and provides new strategies to overcome immune-mediated limitations on efficacy and redosing of GT, which proved effective across two different monogenic diseases and viral vector platforms.</p>