245. Enhancing Factor VIII Expression in Platelets of Hemophilia A Mice Following Intraosseous Delivery of Lentiviral Vectors
Authors: Xuefeng Wang, Meghan J. Lyle, Richard Fu, Carol H. Miao
Journal: Molecular Therapy (2015)

In previous studies, we successfully demonstrated that following intraosseous (IO) infusion of lentiviral vectors (LVs) carrying a transgene encoding human factor VIII variant (hFVIII/N6) driven by a platelet-specific promoter (Gp1bα) into hemophilia A (HemA) mice without preconditioning, FVIII stored in platelet α-granules can partially correct phenotype over 5 months in hemophilia A mice with or without pre-existing inhibitors. In this study, we seek two strategies to further improve factor VIII expression levels in platelets. Firstly, a new human B domain-deleted FVIII variant (BDDFVIII-X10; a kind gift from Dr. Weidong Xiao) which contains a 10-amino acid change in the A1 domain of the FVIII heavy chain was generated. Following hydrodynamic injection of plasmids containing human FVIII variants (BDDFVIII-X10 or hFVIII/N6) driven by a human elongation factor-1 promoter (pEF1α-BDDFVIII-X10 or pEF1α-hFVIII/N6, 50 mg/mouse, n=6) into hemophilia A mice, BDDFVIII-X10 produced a 6-fold increase (532±18% vs 81±25%) in the clotting activity determined by an aPTT assay on day 1. Thus, four LVs containing BDDFVIII-X10 or hFVIII/N6 transgene driven by Gp1bα (G-F8X10-LV or G-F8-LV) or EF1α (E-F8X10-LV or E-F8-LV) promoter were constructed, respectively. Flow cytometry data showed that for human Dami megakaryocyte cells transduced with same dose of LVs, G-F8X10-LV produced a 2-fold increase of hFVIII+Dami cells (23.8% vs 13.4%) compared to G-F8-LV. In 293T cells, E-F8X10-LV produced a 3-fold increase of hFVIII+293T cells (42.6% vs 12.5%) compared to E-F8-LV. These results indicate that BDDFVIII-X10 may serve as a better variant candidate for gene therapy of hemophilia A. Secondly, we further explored the use of pharmacological agents to enhance lentiviral transduction efficiency by either suppressing the initial immune responses against LVs and LV-transduced cells or augmenting the frequency and number of viral entry and integration. The immune competent BL6 mice were pretreated with dexamethasone (Dex, IP injection, 5 mg/kg at -24h, -4h, 4h and 24h, n=9) or combination drugs (Dex, IP injection, 5 mg/kg at -24h, -4h, 4h and 24h; anti-CD8α antibody, IP injection, 4 mg/kg on day -1 and 2.5 mg/kg on day 4, 11, 16 and 21; rapamycin, IO infusion on day 1, 0.25 mg/kg), and IO infusion of GFP-LVs driven by an ubiquitous MND promoter (1.1×108i.f.u./mouse on day 0). On day 7, Dex only and combination drugs + LVs treated mice produced higher numbers of GFP+HSCs in the bone marrow (BM) compared with LV-only treated mice (62.4±5.0% and 50.6±5.7% vs 41.1±12.0%, P=0.003 and 0.06). Similarly, in the long term, higher numbers of GFP+HSCs were observed in BM of Dex only and combination drugs + LVs treated mice compared with LV-only treated mice (10.1±9.2% and 11.7±7.3% vs 6.0±1.6%, P=0.13 and 0.04). Taken together, these results indicate that IO infusion of LVs containing BDDFVIII-X10 into HemA mice pretreated with pharmacological drugs can be used to further enhance and prolong transgene expression levels in platelets for better correction of hemophilia A, in particular in patients with high risk of inhibitory antibody formation.