Correction of Murine Bernard Soulier Syndrome by Lentivirus-Mediated Gene Therapy

Abstract 552

Bernard Soulier Syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib/IX complex. The main treatment for BSS is platelet transfusion but it is often limited to severe bleeding episodes or surgical interventions due to the risk of developing allo-immunization. Ware and colleagues have developed a murine model of BSS by targeting GPIbα (GPIbα^null), and have shown that the BSS phenotype was rescued by transgenic expression of hGPIbα. We have previously reported successful expression of human GPIbα in human megakaryocytes using a lentiviral vector (LV) encoding hGPIbα under the transcriptional control of integrin aIIb promoter (2bIbα). In this study, we examined the efficacy of this strategy for the gene therapy of BSS using GPIbα^null as a murine model of BSS. GPIbα^null hematopoietic stem cells (HSC) transduced with 2bIbα LV were transplanted into lethally irradiated GPIbα^null littermates. After bone marrow (BM) reconstitution, mice were analyzed. The presence of 2bIbα transgene in recipients was confirmed by PCR amplification of white blood cell derived genomic DNA. Flow cytometry demonstrated that 84.5% ± 9.5% (n = 9) of platelets expressed hGPIbα at 6 weeks after transplantation and stable expression was maintained through the entire observation period of 7 months. Immunofluorescent confocal microscopy demonstrated that transgene protein was expressed on the cell surface of transduced platelets. Tail bleeding times were corrected to normal levels in the GPIbα^null recipients who received LV transduced GPIbα^null HSC (2.3 ± 2.9 minutes, n = 9). On the other hand, recipients who received untransduced GPIbα^null HSC exhibited prolonged bleeding times (8.8 ± 2.4 minutes, n = 4) that were similar to GPIbα^null mice. Macrothrombocytopenia improved with significantly increased platelet counts and decreased platelet sizes in LV transduced GPIbα^null HSC recipients compared to untransduced GPIbα^null HSC recipients (platelet counts; 4.9 ± 1.3×10⁵/μ l, n = 9 vs. 1.8 ± 0.1×10⁵/μ l, n = 4, and mean platelet volume; 6.9 ± 0.7 fL, n = 9 vs. 9.3 ± 0.1 fL, n = 4, respectively). As expected, expression levels of hGPIbα correlated with platelet counts and inversely correlated with the platelet size. Immunoprecipitation followed by Western blot analysis showed that hGPIbα expressed on platelets associated with endogenous murine GPIbβ and GPIX. No antibody to hGPIbα was detected in these recipients. Furthermore, BM mononuclear cells from the primary recipients were transplanted into the secondary GPIbα^null recipients and the results showed sustained expression of hGPIbα leading to the correction of bleeding phenotype as well as macrothrombocytopenia. These results demonstrate that lentivirus mediated gene transfer can provide sustained phenotypic correction of murine BSS, indicating that this approach may be a promising strategy for gene therapy of BSS in human.