Akira Ishiwata

Efficient expression of a transgene in platelets using simian immunodeficiency virus-based vector harboring glycoprotein Ibα promoter: in vivo model for platelet-targeting gene therapy

Platelets release several mediators that modify vascular integrity and hemostasis. In the present study, we developed a technique for efficient transgene expression in platelets in vivo and examined whether this targeted‐gene‐product delivery system using a platelet release reaction could be exploited for clinical applications. Analysis of luciferase reporter gene constructs driven by platelet‐specific promoters (the GPIIb, GPIbα, and GPVI) revealed that the GPIbα promoter was the most potent in the megakaryoblastic cell line UT‐7/TPO and human CD34+‐derived megakaryocytes. Transduction of UT‐7/TPO;CD34+‐derived megakaryocytes; and c‐Kit+, ScaI+, and Lineage− (KSL) murine hematopoietic stem cells with a simian immunodeficiency virus (SIV)‐based lentiviral vector carrying eGFP resulted in efficient, dose‐dependent expression of eGFP, and the GPIbα promoter seemed to bestow megakaryocytic‐specific expression. Transplantation of KSL cells transduced with SIV vector containing eGFP into mice showed that there was preferable expression of eGFP in platelets driven by the GPIbα promoter [7–11% for the cytomeglovirus (CMV) promoter, 16–27% for the GPIbα promoter]. Furthermore, transplantation of ex vivo‐transduced KSL cells by SIV vector carrying human factorVIII (hFVIII) driven by the GPIbα promoter induced the production of detectable transcripts of the hFVIII gene and the hFVIII antigen in bone marrow and spleen for at least 90 days and partially corrected the hemophilia A phenotype. Platelet‐targeting gene therapy using SIV vectors appears to be promising for gene therapy approaches toward not only inherited platelet diseases but also other hemorrhagic disorders such as hemophilia A.—Ohmori, T., Mimuro, J., Takano, K., Madoiwa, S., Kashiwakura, Y., Ishiwata, A., Niimura, M., Mitomo, K., Tabata, T., Hasegawa, M., Ozawa, K., Sakata, Y. Efficient expression of a transgene in platelets using simian immunodeficiency virus‐based vector harboring glycoprotein Ibα promoter: in vivo model for platelet‐targeting gene therapy. FASEB J. 20, E769–E779 (2006)

Minimizing the inhibitory effect of neutralizing antibody for efficient gene expression in the liver with adeno-associated virus 8 vectors.

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 ± 2.10-10.1 ± 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 ± 2.59-12.68 ± 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 ± 1.06-9.0 ± 2.37%) in the presence of NAbs (14-56× dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.

Liver-restricted expression of the canine factor VIII gene facilitates prevention of inhibitor formation in factor VIII-deficient mice.

Gene therapy for hemophilia A with adeno‐associated virus (AAV) vectors involves difficulties in the efficient expression of factor VIII (FVIII) and in antibody formation against transgene‐derived FVIII.

Phenotypic Correction of Hemophilia A by Ectopic Expression of Activated Factor VII in Platelets

Platelets are receiving much attention as novel target cells to secrete a coagulation factor for hemophilia gene therapy. In order to extend the application of platelet-directed gene therapy, we examined whether ectopic expression of activated factor VII (FVIIa) in platelets would result in an efficient bypass therapy to induce sufficient thrombin generation on platelet surfaces in mice with hemophilia A. Transduction of bone marrow cells with a simian immunodeficiency virus (SIV)-based lentiviral vector harboring the platelet-specific GPIb alpha promoter resulted in efficient transgene expression in platelets. FVIIa antigen was expressed in platelets by this SIV system; FVII transgene products were found to localize in the cytoplasm and translocate toward the sub-membrane zone and cell surface after activation. Although FVII antigen levels in platelets did not reach the therapeutic levels seen with FVIIa infusion therapy, whole-blood coagulation, as assessed by thromboelastography, was significantly improved in mice with hemophilia A. Further, we observed correction of the bleeding phenotype in mice with hemophilia A after transplantation, even in the presence of FVIII-neutralizing antibodies. Our results demonstrate that FVIIa-expressing platelets can strengthen hemostatic function and may be useful in treating hemophilia and other inherited bleeding disorders. These findings are comparable to the proven therapeutic effects of FVIIa infusion.

Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice.

An Erratum has been published for this article in Journal of Gene Medicine 7(6), 2005, 836.

Porcine Model of Hemophilia A

Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.

Degradation of cross-linked fibrin by leukocyte elastase as alternative pathway for plasmin-mediated fibrinolysis in sepsis-induced disseminated intravascular coagulation

An alternative pathway for fibrinolysis that comprises leukocyte elastase and its interaction with the plasminogen activator-plasmin system has been suggested. Plasma levels of cross-linked fibrin degradation product by leukocyte elastase (e-XDP) were significantly increased in patients with sepsis induced disseminated intravascular coagulation (DIC) compared with healthy subjects (18.6±19.9 vs 0.58±0.47U/mL, p<0.001). Twenty seven unique spots were identified from e-XDP dominant patients by immune-purification and two-dimensional difference gel electrophoresis, and they contained fibrinogen Bβ-chain derived fragments Bβ Asp-164, Ser-200, Gln-301, Ala-354, Ile-484 and γ-chain derivatives γ Val-274 at their amino-termini by acquired and processed tandem mass spectrometer. The Sequential Organ Failure Assessment Scores in patients with e-XDPs levels 3-10U/mL were significantly lower than those with e-XDPs levels -3U/mL, 10-30U/mL, and 30- U/mL. The adjusted odds for 28-day mortality rate in patients with e-XDP levels less than 3U/mL (hazard ratio, 4.432; 95% CI, 1.557-12.615 [p=0.005]) were significantly higher than those in patients with e-XDP levels of 3-10U/mL. These data suggest that leukocyte elastase might contribute to the degradation of cross-linked fibrin in sepsis-induced DIC.

Induction of factor VIII‐specific unresponsiveness by intrathymic factor VIII injection in murine hemophilia A

Summary.  Background: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen‐specific immune tolerance by interferon‐γ (IFN‐γ)‐dependent T‐cell anergy in hemophilia A mice. Objective: The thymus plays crucial roles in self‐tolerance, with negative selection of self‐reactive effector T cells and positive selection of self‐reactive regulatory T cells. We investigated the possibility of the induction of antigen‐specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. Methods: Hemophilia A mice were injected with recombinant FVIII into the thymus under real‐time high‐resolution image guidance. Results: Anti‐FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 ± 2.3 vs. 122.5 ± 27.6 BU mL−1, respectively, P = 0.00078). The CD4+ T cells from thymic‐injected mice could not proliferate or produce interleukin (IL)‐2, IL‐12 and IFN‐γ in response to FVIII. The CD4+CD25+ T cells generated from thymic‐treated mice but not from naïve mice efficiently suppressed the in vitro proliferative response of CD4+ T cells and blocked the in vivo development of anti‐FVIII antibodies in the adoptive transfer. Conclusion: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII‐specific regulatory T cells.

Silencing of a Targeted Protein in In Vivo Platelets Using a Lentiviral Vector Delivering Short Hairpin RNA Sequence

Objective— Because platelets are anucleate cells having a limited life span, direct gene manipulation cannot in principle be used to investigate the involvement of a specific signal transduction pathway in platelet activation. In this study, we examined whether the expression of a short hairpin RNA (shRNA) sequence in hematopoietic stem cells is maintained during megakaryocyte differentiation, thus resulting in inhibition of targeted protein in platelets. Methods and Results— To identify platelets derived from transduced stem cells, we generated a lentiviral vector that simultaneously expresses the shRNA sequence driven by the U6 promoter and GFP under the control of the glycoprotein (GP) Ib&agr; promoter. Transplantation of mouse bone marrow cells transduced with the vector facilitated specifically mark platelets derived from the transduced cells. Transplantation of cells transduced with shRNA sequence targeting integrin &agr;IIb caused a significant reduction of integrin &agr;IIb&bgr;3 (&agr;IIb&bgr;3) expression in GFP-positive platelets. It also inhibited &agr;IIb&bgr;3 activation assessed by the binding of JON/A, an antibody that recognizes activated &agr;IIb&bgr;3. Talin-1 silencing by the same method resulted in normal &agr;IIb&bgr;3 expression but deficient inside-out &agr;IIb&bgr;3 signaling. Conclusions— shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. This method facilitates functional analysis of targeted protein in platelet activation.

Vinculin Is Indispensable for Repopulation by Hematopoietic Stem Cells, Independent of Integrin Function

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function, little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit+Sca1+Lin− HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages, to migrate toward stromal cell-derived factor 1α, and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However, the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit+Sca1+Lin− HSCs. In contrast, adhesion to the extracellular matrix was inhibited by silencing of talin-1, but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity, independent of integrin functions.