5. Development of Enhancing Intraosseous Delivery Efficiency of LV-Factor VIII Variants in Platelets of Hemophilia A Mice
Authors: Xuefeng Wang, Richard Y. Fu, Carol H. Miao
Journal: Molecular Therapy (2016)

Introduction: Our previous studies demonstrated that human factor VIII (FVIII) specifically expressed in megakaryocytes and then stored in platelets of Hemophilia A (HemA) mice can partially correct their phenotype over 5 months in mice with or without pre-existing inhibitors. This was achieved by intraosseous (IO) delivery of lentiviral vectors (LVs) carrying a transgene encoding human FVIII variant (BDDhFVIII/N6; abbreviated as F8) driven by a megakaryocyte-specific promoter (Gp1bα) without preconditioning as required in ex vivo gene therapy. Methods: In this study, we aimed at enhancing transgene expression by two strategies. One was to enhance LV transduction efficiency by suppressing the innate and adaptive immune responses against LVs and LV-transduced cells using pharmacological agents. The other was to improve FVIII gene expression by incorporating a new human FVIII variant, F8X10K12 (a 10-amino acid change in the A1 domain and a 12-amino acid change in the light chain; a kind gift from Dr. Weidong Xiao). Results: First, the immune competent C57BL6 mice were pretreated with both dexamethasone (Dex) (IP, 5 mg/kg at -24h, -4h, 4h and 24h) and anti-CD8α monoclonal antibody (mAb) (IP, 4 mg/kg on day -1, 4, 11, 16 and 21), or Dex only. IO infusion of GFP-LVs (1.1×108 i.f.u./mouse) driven by a ubiquitous MND promoter was performed on day 0. On day 7, Dex only and combination drugs + LVs treated mice (n=3) produced higher numbers of GFP+ total bone marrow cells (17.7±3.5% and 11.8±2.1% vs 6.9±3.1%, P=0.0001 and 0.005) and GFP+Lineage-Sca1+cKit+ HSCs (55.5±3.1% and 48.3±6.1% vs 44.4±17.2%, P=0.1 and 0.31) compared with LV-only treated mice (n=3). Most importantly, in the long term, higher numbers of GFP+ cells (2.4±0.4% vs 0.5±0.1%, P<0.001) in the total bone marrow and GFP+HSCs (10.7±3.3% vs 2.6±0.6%, P<0.001) were observed in combination drugs + LVs treated mice (n=3) compared with LV-only treated mice on day 160 after LV infusion (n=3), which was confirmed by higher LV copy number in bone marrow cells of drugs + LVs treated mice. Second, we tested the FVIII expression levels from two human FVIII variants in HemA mice by hydrodynamic injection of plasmids driven by a human elongation factor-1 promoter (pEF1α-F8X10K12 or pEF1α-F8, 50 µg/mouse, n=8), respectively. Compared with F8, F8X10K12 produced a 25-fold increase (147±27% vs 3,734±477%) in the clotting activity determined by an aPTT assay on day 4 post injection. Then two LVs containing F8X10K12 or F8 transgene driven by EF1α promoter (E-F8X10K12-LV or E-F8-LV) were constructed and used to transduce 293T cells, respectively. Flow cytometry data showed that E-F8X10K12-LV produced a significant increase of hFVIII+293T cells (77.8% vs 15%) and MFI (795 vs 541) compared to E-F8-LV at the same doses. These results indicated that F8X10K12 may further enhance FVIII gene expression in platelets for more effective therapy. LVs containing F8X10K12 or F8 transgene driven by Gp1bα promoter (G-F8X10K12-LV or G-F8-LV) were subsequently generated and were intraosseously delivered into HemA mice to test the FVIII efficiency in platelets by ELISA, thrombin generation assay and tail clipping. Conclusion: We found that administration of Dex that efficiently inhibited initial innate immune responses to LVs in vivo combined with anti-CD8α mAb that depleted subsequent cytotoxic CD8+ T cells improved the transduction efficiency of LVs and persistence of transduced cells, leading to over 10% GFP+HSCs in treated mice up to 160 days. In addition, a new FVIII variant, F8X10K12, can significantly enhance FVIII expression in mice following hydrodynamic injection of plasmids and in LV-transduced cells. Taken together, IO infusion of G-F8X10K12-LV into HemA mice pretreated with Dex and antiCD8α mAb can be used to further enhance and prolong transgene efficiency in platelets for effective correction of hemophilia A.