522. Targeting FVIII-Expression To Liver Sinusoidal Cells By Lentiviral Vectors Corrects the Bleeding Phenotype in Hemophilia A Overcoming Immunological Responses

Hemophilia A (HA) is an X-linked bleeding disorder due to mutations in clotting factor (F) VIII gene. To date the treatment for preventing major bleeding episodes is represented by replacement therapy with recombinant or plasma-derived FVIII. The two major concerns are high cost and development of FVIII neutralizing antibodies in 20-30% of patients.Several studies on gene transfer by direct injection of LV for HA have been recently published. Many efforts were focused on the improvement of LV, to obtain a selective targeting of transgene expression, or on the production of several bioengineered FVIII, in order to overcome some of the issues related to FVIII expression in HA animal models. However, in most cases, the immune responses associated with FVIII remain the major obstacle.We prepared LVs containing the B-domain deleted (BDD) hFVIII under the control of PGK, VEC or CD11b promoters with or without the addition of the miRTs used for initial GFP expression studies, and we then injected HA mice with 109 TU/mouse of these LVs (3 mice for LV PGK-hFVIII ±42; 4-9 mice for the other vectors) and assessed FVIII activity by aPTT assay.All mice injected with LV-VEC-hFVIII ± miRTs and LV-CD11b-hFVIII ± miRTs showed a FVIII activity between 3.5 and 5% one week after injection, while HA mice injected with LV-PGK-hFVIII± 42 showed a FVIII activity £1%. Moreover, starting from 2 weeks after LVs injection we evaluated the presence of anti-FVIII antibodies by a direct ELISA. We detected the presence of anti-FVIII antibodies in the plasma of mice injected with LV-PGK-hFVIII±miRT-142 1 month after LV injection. Interestingly, the antibody titer was significantly lower in mice injected with LV-PGK-hFVIII-miRT-142-3p. In all mice injected with LV-VEC-hFVIII±miRT-122-142-3pwe detected hFVIII activity by aPTT assay up to 52 weeks after injection without production of anti-FVIII antibodies. HA mice injected LV-CD11b-hFVIII±miRT-126 showed hFVIII activity up to 52 w as well; interestingly, 60% of mice injected with LV-CD11b-hFVIII produced anti-FVIII antibodies 10-16 weeks after LV injection, while no anti-FVIII antibodies were detected in plasma of injected mice with LV-CD11b-hFVIII-miRT-126.Genomic analysis on liver samples from mice 24 w after injection of LV-VEC-hFVIII±miRT-122-142-3p and LV-CD11b-hFVIII±miRT-126 demonstrated the presence of LV sequence integrated in the genome of injected mice. Immunofluorescence on liver sections showed that LSECs and KCs were positive for hFVIII. Next, to assess whether EC, in particular LSECs, are able to induce immunotolerance, we immunized mice with Refacto. Mice producing anti-FVIII Ab were then injected with 109 TU of LV-VEC-hFVIII-miRT-122-142-3p. We detected hFVIII activity in all injected mice and, noteworthy, antibody titer decreased over time in the plasma of these mice.In conclusion, LV expressing FVIII under the control of VEC or CD11b promoters combined with miRTs combinations were able to overcome FVIII off-target expression limiting immune responses and providing phenotypic correction in treated HA mice with FVIII expression by sinusoidal cells.