These results demonstrate that lentivirus-mediated gene transfer can provide sustained phenotypic correction of murine BSS, indicating that this approach may be a encouraging strategy for gene therapy of BSS patients

These results demonstrate that lentivirus-mediated gene transfer can provide sustained phenotypic correction of murine BSS, indicating that this approach may be a encouraging strategy for gene therapy of BSS patients. Introduction The BernardCSoulier syndrome (BSS) is an autosomal recessive disease characterized by thrombocytopenia, enlarged platelets, and bleeding symptoms.1,2 BSS is caused by mutations in one of the three genes encoding the glycoprotein (GP) Ib-IX-V complexunder transcriptional control of the integrin IIb promoter that expressed hGPIb efficiently inside a lineage-specific manner.19 Ware and colleagues have developed a murine model of BSS by disrupting the gene (GPIbnull), and have shown the BSS phenotype was rescued by transgenic expression of hGPIb.20 In the present study, we examined the effectiveness of 2bIb LV-mediated bone marrow (BM) transduction and syngeneic transplantation for the gene therapy of BSS using a GPIbnull murine model of BSS. Results Manifestation of hGPIb in GPIbnull mice We had previously constructed a 2bIb LV vector that expresses hGPIb under the control of the integrin IIb promoter and confirmed efficient manifestation inside a megakaryocytic cell collection (Dami) and human being CD34+ cells.19 To assess the use of our 2bIb LV for gene therapy of BSS, HSC isolated from GPIbnull mice were transduced and transplanted into lethally irradiated GPIbnull littermates. transplants showed sustained manifestation of hGPIb with related phenotypic correction. Antibody response to hGPIb was recorded in 1 of 17 SULF1 total recipient mice but was tolerated without any further treatment. These results demonstrate that lentivirus-mediated gene transfer can provide sustained phenotypic correction SYP-5 of murine BSS, indicating that this approach may be a encouraging strategy for gene therapy of BSS individuals. Intro The BernardCSoulier syndrome (BSS) is an autosomal recessive disease characterized by thrombocytopenia, enlarged platelets, and bleeding symptoms.1,2 BSS is caused by mutations in one of the three genes encoding the glycoprotein (GP) Ib-IX-V complexunder transcriptional control of the integrin IIb promoter that expressed hGPIb efficiently inside a lineage-specific SYP-5 manner.19 Ware and colleagues have developed a murine model of BSS by disrupting the gene (GPIbnull), and have shown the BSS phenotype was rescued by transgenic expression of hGPIb.20 In the present study, SYP-5 we examined the effectiveness of 2bIb LV-mediated bone marrow (BM) transduction and syngeneic transplantation for the gene therapy of BSS using a GPIbnull murine model of BSS. Results Manifestation of hGPIb in GPIbnull SYP-5 mice We had previously constructed a 2bIb LV vector that expresses hGPIb under the control of the integrin IIb promoter and confirmed efficient manifestation inside a megakaryocytic cell collection (Dami) and human being CD34+ cells.19 To assess the use of our 2bIb LV for gene therapy of BSS, HSC isolated from GPIbnull mice were transduced and transplanted into lethally irradiated GPIbnull littermates. Recipients were analyzed after BM reconstitution and the presence of 2bIb transgene in recipients was confirmed by PCR amplification of peripheral white blood cell-derived genomic DNA (Number 1a). All GPIbnull mice that received LV-transduced HSC were positive for 2bIb transgene. The average copy quantity of 2bIb proviral DNA was 0.42 0.31 copies per white blood cell in transduced recipients. Manifestation of the hGPIb transgene protein in platelets was confirmed by immunofluorescent confocal microscopy. Most of the platelets were positively stained for hGPIb in 2bIb LV-transduced HSC recipients (Number 1b). The merged image demonstrates the hGPIb protein did not colocalize with the endogenous -granule protein, VWF, but was indicated within the plasma membrane of transduced platelets. Open in a separate window Number 1 Genetic and manifestation analysis of 2bIbLV-transduced bone marrow transplantation (BMT) recipients. (a) PCR analysis of BMT recipients shows the presence of transgene in recipients. Genomic DNA was prepared from main (1) and secondary (2) 2bIb lentiviral vector (LV) transduced hematopoietic stem cells (HSC) recipients. GPIbnull and C57BL/6J wild-type mice were used as settings. 2bIb LV plasmid DNA was used like a positive control for human being GPIb (hGPIb). Absence of mGPIb PCR product confirmed the GPIbnull background. The gene was used as an internal control. PCR product sizes; hGPIb (458 bp), mGPIb (486 bp), and Vwf (727 bp). (b) Immunofluorescent staining of mouse platelets. Platelets were isolated from GPIbnull mice that received 2bF8 LV-transduced HSC (top panel) and untransduced GPIbnull control mice (lower panel) and stained for hGPIb (green) and murine VWF (reddish). Nonspecific isotype-matched main antibodies were used to assess background staining (data not shown). Pub = 10?m. The percentage of platelets that indicated hGPIb was analyzed by circulation cytometry and ranged from ~70 to 90% (Number 2a). Normally, 84.5 9.5% (= 9) of total platelets were expressing hGPIb at 6 weeks after transplantation in 2bIb LV-transduced HSC recipients and stable expression was maintained through the entire observation period of 7 months (Figure 2b). The integrin IIb gene promoter that we used in our LV vector offers previously been characterized and shown to induce platelet-specific manifestation and = 9) was plotted at each time point. Untransduced settings (= 4) were analyzed in parallel each time. Data is indicated as the mean SD. (c) Platelet-specific manifestation of.