Targeting FVIII Expression to Human Platelets Corrects the Hemophilic Phenotype in An Immunocompromised Hemophilia A Mouse Model Transplanted with Genetically Manipulated Human Cord Blood Stem Cells

Abstract 20 Hemophilia A is a strong candidate for gene therapy because the therapeutic window is broad and a low plasma concentration of FVIII is therapeutic. However, the results of clinical trials so far have been disappointing due to either low expression or immune response problems. Our previous studies have demonstrated that lentivirus-mediated platelet-specific expression of FVIII (2bF8) in mouse platelets can ameliorate the bleeding phenotype in murine hemophilia A even in the presence of inhibitory antibodies. Since our ultimate goal is to express FVIII in human hemophilia patients9 platelets, we wanted to explore whether lentivirus-mediated gene transfer can efficiently introduce therapeutic levels of FVIII in human platelets and whether this human platelet-derived FVIII can correct the hemophilic phenotype. Human platelet-derived FVIII expression was introduced by 2bF8 lentivirus (LV)-mediated transduction of human cord blood (hCB) CD34 + cells followed by xeno-transplantation (hCBT) into immunocompromised NOD-scid IL2r-gamma knockout (NSG) mice. After bone marrow reconstitution, recipients were analyzed by flow cytometry, PCR, platelet lysate FVIII:C assay, immunogold-staining electron microscopy (EM), and non-restrictive LAM-PCR (nrLAM-PCR). About 30–67% of platelets in NSG/CBT mice were of human origin 3 weeks after transplantation, dropping to 3–18% at 5 weeks when 47–98% white blood cells (WBC) were of human origin. Expression of the 2bF8 protein product was detected in all recipients that received 2bF8 LV-transduced hCB cells, indicating viable engraftment of human CD34 + cells that were genetically modified with the 2bF8 LV transfer vector. Functional platelet-FVIII activity levels in the transduced mice were 0.74 ± 0.45 mU/10 8 total platelets (n = 6), which corresponded to 17.77 ± 10.36 mU/10 8 human platelets when converted to human platelets according to chimerism levels measured by FACS. EM determined that transgenic FVIII was colocalized with endogenous VWF in 2bF8 LV-transduced human platelets from hCBT recipients. nrLAM-PCR analysis showed that no identified insertion sites of 2bF8 LV were located in areas listed in the Retrovirus Tagged Proto-oncogene in human hematopoiesis database. To explore whether 2bF8 LV-transduced human platelets can ameliorate the bleeding phenotype of hemophilia A, we generated an immunocompromised hemophilia A mouse (SG/FVIII null ) model by crossing NSG mice into FVIII null background and xeno-transplanted 2bF8 LV-transduced hCB CD34 + cells into these mice. We found that human platelet chimerism in recipients varied markedly with loss of NOD phenotype during breeding, while WBC chimerism was not affected. PCR analysis of 2bF8 proviral DNA confirmed viable 2bF8 LV-transduced human hematopoietic engraftment. FVIII:C was detected in platelet lysates with levels of 0.57 ± 0.41 mU/10 8 total platelets (18.30 ± 9.65 mU/10 8 human platelets (n = 11)). Four weeks after transplantation, all recipients (n = 7) that received 2bF8 LV-transduced hCB CD34 + cells survived tail clipping if animals had greater than 2% (3–57%) of platelets derived from 2bF8 LV-transduced hCB CD34 + cells, while 4 of 6 survived when human platelets were between 0.3% and 2%. Reduced survival in the latter group is presumably due to the limited number of human platelets containing FVIII. Rotational Thromboelastometry (TEG-like) analysis of whole blood confirmed that hemostasis was improved in SG/FVIII null mice that received 2bF8 LV-transduced hCB CD34 + cells. Our results demonstrate that 2bF8 LV can efficiently introduce FVIII expression in human platelets and that human platelet-derived FVIII can improve hemostasis in hemophilia A mice, indicating that lentivirus-mediated platelet-derived FVIII gene therapy may be a promising approach for the treatment of human patients with hemophilia A. Disclosures: No relevant conflicts of interest to declare.