Development of Direct Intra-Bone Marrow Gene Transfer of Lentiviral Vectors Containing a B-Domain Variant of Human Factor VIII for Hemophilia A Treatment

Abstract 4710

Lentiviral vectors (LVs) directly delivered to the bone marrow by intra-bone marrow (iBM) injection can efficiently transduce hematopoietic stem cells (HSCs) for treating genetic blood disorders. Our previous study showed that FVIII activity in the plasma of hemophilia A (HemA) mice achieved nearly normal levels one week after being treated with iBM injection of LVs containing a B-domain variant human factor VIII (hFVIII/N6) gene driven by a human elongation factor-1 promoter (E-LV). However, plasma FVIII activity fell very quickly with concomitant generation of anti-FVIII antibodies. In this study, to prevent anti-FVIII immune responses in the treated HemA mice, we treated three groups of mice with intraperitoneal injection of 200 μL of immunomodulatory protocols including IL-2/IL-2 mAb complexes (IL-2 Complex, 1 μg IL-2 mixed with 5 μg anti-IL-2 mAb per mouse on day −5, −4, and −3), anti-mouse CD3ε mAb (20 μg per mouse on day −2, −1, 0, 1 and 2), and rabbit anti-mouse thymocyte IgG (ATG, 0.6 mg per mouse on day −1 and 0), respectively. The mice were treated with 5 μL E-LV (7 × 10⁷ TU/mL) on day 0. The peak plasma FVIII levels in 0/4 E-LV only treated mice, 3/5 IL-2 Complex pretreated mice, 2/3 anti-CD3ε pretreated mice and 4/5 ATS pretreated mice showed up one month after treatment. 2/4 E-LV only treated mice, 2/5 IL-2 Complex pretreated mice, 1/3 anti-CD3ε pretreated mice and 3/5 ATS pretreated mice generated anti-FVIII antibodies two months after treatment. These data indicated that all three immunomodulatory protocols protected E-LV transduced cells from eradication mediated by cytotoxic T cells in the early stage after treatment; however these protocols did not prevent the production of anti-FVIII antibodies at later time points. Next, we employed 10 HemA/Foxp3-Tg mice in which all T cells over-expressed Foxp3 as an immunosuppressive mouse model and treated them with low (5 μL) or high (25 μL) dosage of E-LVs, respectively. None of the 10 treated HemA/Foxp3-Tg produced anti-FVIII antibodies however plasma FVIII levels in all 10 mice fell to low levels one month after treatment. Thus, increase of regulatory T cells could not totally inhibit cytotoxic T cells to lyse transduced cells, whereas they prevented anti-FVIII antibody production. In order to examine whether LV-transduced cells were destroyed in the early stage post-injection, we treated HemA mice with 3 μL of LVs (4.6 × 10¹⁰ TU/mL) containing GFP gene driven by a modified myeloid proliferative sarcoma virus (MND) promoter and tracked GFP expression in bone marrow cells overtime. The percentage of GFP positive cells reached highest on day 15 post-injection, and then fell quickly afterwards, while GFP positive cells (0.04% of total bone marrow cells) were still detectable on day 94. Therefore, in order to achieve persistent transgene expression of hFVIII in the HemA mice treated with intra-bone marrow injection of E-LVs, cytotoxic-T-cell-mediated elimination of the transduced cells needs to be inhibited in the early stage and formation of anti-FVIII antibody can be eliminated by increasing FVIII-specific Treg cell populations.