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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5161fjev1 20/9/1522    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ohmori, T. Articles by Sakata, Y. Search for Related Content PubMed PubMed Citation Articles by Ohmori, T. Articles by Sakata, Y.

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

Tsukasa Ohmori^*, Jun Mimuro^*,, Katsuhiro Takano^*, Seiji Madoiwa^*,, Yuji Kashiwakura^*, Akira Ishiwata^*, Masanori Niimura^*, Katsuyuki Mitomo, Toshiaki Tabata, Mamoru Hasegawa, Keiya Ozawa^, and Yoichi Sakata^*,,1

^* Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan;

Hematology Division of Department of Medicine, Jichi Medical School, Tochigi, Japan;

DNAVEC Corp., Ibaraki, Japan; and

Research Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan

¹Correspondence: Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan. E-mail: yoisaka{at}jichi.ac.jp

SPECIFIC AIMS

Platelets release a number of mediators that modify vascular integrity and hemostasis. The goals of this work were 1) to develop a technique for efficient transgene expression in platelets in vivo; and 2) to examine whether this targeted-gene-product delivery system using the platelet release reaction was directly applicable to gene therapy for coagulation factor deficiency hemophilia A.

PRINCIPAL FINDINGS

1. Comparison of luciferase reporter expression driven by platelet-specific promoters
We first compared the promoter activities of three platelet-specific genes, Glycoprotein (GP) IIb, GPIb, and GPVI, in megakaryocytic cells. The GPIb promoter directed the most powerful expression of luciferase in UT-7/TPO cells, a megakaryoblastic cell line, and CD34⁺-derived megakaryocytes. The platelet-specific promoters drove less reporter gene compared with SV40/enhancer in endothelial cells, smooth muscle cells, and other hematopoietic cell lines.

2. Efficient expression of transgenes in platelets in vivo
We constructed simian immunodeficiency virus (SIV)-based lentiviral vectors containing the eGFP gene under the control of either the cytomegalovirus [cytomeglovirus (CMV)] promoter (SIV-CMV-eGFP), GPIb promoter (SIV-GPIb-eGFP), GPIIb promoter, or GPVI promoter. Transduction of CD34⁺-derived megakaryocytes with the SIV-based lentiviral vectors resulted in efficient, dose-dependent expression of eGFP, and the GPIb promoter seemed to bestow megakaryocytic-specific expression. We selected the GPIb promoter as the platelet-specific promoter for in vivo experiments, because the promoter activity of GPIb was the strongest in megakaryocytes and the promoter drove in the later phase of megakaryopoiesis. We next optimized the transduction protocol of c-Kit⁺, ScaI⁺, and Lineage^– (KSL) murine hematopoietic stem cells using SIV-CMV-eGFP. The transduction efficiency of eGFP in cultured KSL cells reached 60–80%. The plateau value of transduction was observed with a multiplicity of infection (MOI) of 10–30.

To compare the specificity of the CMV and GPIb promoters and to assess eGFP transduction by SIV vectors in vivo, KSL cells transduced with SIV-CMV-eGFP or SIV-GPIb-eGFP were transplanted to recipient mice (Ly5.2). One hundred thousand cultured KSL cells (Ly5.1) transduced with SIV-CMV-eGFP or SIV-GPIb-eGFP (MOI of 30) were transplanted together with 5 x 10⁵ competitor cells (Ly5.2) after lethal -irradiation (9.5 Gy). When KSL cells transduced with SIV-CMV-eGFP were transplanted, eGFP expression was observed in 35–45% of CD45⁺ cells and 7–11% of platelets in peripheral blood (Fig. 1 A and B). Interestingly, transduction of SIV vector harboring the GPIb promoter would be more likely to result in efficient gene marking to platelets (16–27%; Fig. 1A and B ). We next analyzed eGFP expression of bone marrow cells from transplanted mice. Whereas eGFP was expressed in the lineage cells of mice that received KSL cells transduced with SIV-CMV-eGFP, the GPIb promoter drove a marginal eGFP expression in these cell lineages, confirming the specificity of its activity in megakaryocytes and platelets (Fig. 1C ). Next, we performed second bone marrow transplantations using marrow cells obtained from mice that had been transplanted 4 months earlier. As shown in Fig. 1D , eGFP expression driven by the CMV and GPIb promoters in hematopoietic cells was sustained after the second stem cell transplantation.

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of promoter differences on eGFP expression in blood cells in vivo. Cultured KSL cells were transduced with SIV-CMV-eGFP or SIV-GPIb-eGFP at a MOI of 30. Each irradiated mouse received 100,000 transduced cells together with 5 x 105 unfractionated whole marrow cells. A) Representative flow cytometry analyses of eGFP-positive cells in CD45+ lymphocytes and granulocytes, red blood cells (RBCs), and platelets in peripheral blood are shown. B) Percentages of eGFP-positive cells in CD45+ cells (left) and platelets (right) 14, 30, and 60 days after transplantation are shown. Columns and error bars are mean ± SD (n=5 per group). C) 60 days post-transplantation, bone marrow cells were stained using antibodies to detect B lymphocytes (B220), T lymphocytes (CD3), granulocytes (Gr1), macrophages (CD11b), and erythroblasts (TER119). GFP-positive cells in each lineage cells are measured by flow cytometry. Data represent 3 experiments. D) Flow cytometric analyses of CD45+ cells, RBCs, and platelets in peripheral blood obtained from mice 30 days after second bone marrow transplantation. Columns and error bars are mean ± SD (n=5 per group).

3. Phenotypic correction of hemophilia A mice (factor VIII-deficient mice)
To determine whether platelet-directed gene therapy enables the sustained expression of coagulation factor VIII (FVIII), we constructed two SIV-based vectors containing the human FVIII (hFVIII) cDNA under the control of either CMV (SIV-CMV-hFVIII) or GPIb promoter (SIV-GPIb-hFVIII). We first analyzed the presence of the hFVIII gene transcripts in organs of the transplanted recipients 3 months after transplantation. Real-time quantitative reverse transcriptase-polymerase chain reaction revealed that bone marrow and spleen are the major expression sites in mice transplanted with KSL cells transduced with SIV vectors. In accordance with the data on hFVIII transcripts, hFVIII molecules were immunohistochemically detected in bone marrow and the spleen in both types of transduced mice.

We finally evaluated whether platelet-specific gene transduction using SIV-GPIb-hFVIII resulted in phenotypic correction of FVIII-deficient hemophilia A mice. The plasma hFVIII antigen concentration without or with platelet activation was measured in transplanted FVIII-deficient mice at 30 and 60 days after transplantation. We detected FVIII activity in transplanted mice; 1–2% correction was noted in the plasma of mice transplanted with KSL cells transduced with SIV-GPIb-hFVIII (Fig. 2 A). When platelets were stimulated with collagen and PMA, the plasma FVIII concentration increased to 2–3.5% (Fig. 2A ). The mortality rate after tail clipping was significantly improved in transduced mice (Fig. 2B ). Furthermore, ectopically expressed hFVIII levels did not attenuate with time, and the appearance of inhibitor against hFVIII was not detected in mice transplanted with KSL cells transduced with SIV-GPIb-hFVIII at day 60 after transplantation.

View larger version (12K): [in this window] [in a new window]   Figure 2. Phenotypic correction of hemophilia A mice by platelet-targeting gene delivery. A) Blood from FVIII-deficient mice transplanted with KSL cells transduced with SIV-GPIb-hFVIII was stimulated without or with 50 µg/ml of collagen and 1 µM PMA for 15 min. After centrifugation, platelet-poor plasma was obtained, and hFVIII antigen levels were measured by ELISA. Columns and error bars are mean ± SD (n=4 per group). B) Mortality rate within 24 h after tail clipping in mice transplanted with KSL cells transduced with control or SIV-GPIb-hFVIII (n=10 for control; n=8 for GPIb). Mortality rate was statistically evaluated by a 2 test.

CONCLUSION AND SIGNIFICANCE

In this study, we examined gene transduction of platelets and megakaryocytes using an SIV lentiviral vector harboring a platelet-specific promoter in vivo. Since the strategy of using platelets as potential targets for producers of transgene products has already been proposed in transgenic mice, it was possible to apply this strategy to correct coagulation abnormalities including hemophilia A by efficient platelet-directed gene transduction in vivo. In our system, the transduction of hematopoietic stem cells with the SIV lentiviral vector resulted in expression of the transgene in 20% of platelets, and ectopically expressed FVIII in platelets resulted in phenotypic correction of hemophilia A mice.

Blood platelets, the principal cells responsible for primary hemostasis at the site of vascular injury, activated platelets aggregate and release several mediators that modify vascular integrity and hemostasis. Taking advantage of the platelet-release reaction as a delivery system for a specific factor would be a reasonable approach for treatment of individuals deficient in the factor, because it provides a way to enhance the local concentration of target substances at the site of vascular injury, while minimizing the influence of plasma proteins that may inhibit their activities (Fig. 3 ).

View larger version (15K): [in this window] [in a new window]   Figure 3. Expected advantages of platelet-directed gene therapy.

Megakaryocytes have a finite life span; therefore, hematopoietic stem cells are a more practical target than megakaryocytes for genetic transfer to establish long-term expression of a target protein in platelets. Because lentiviruses are capable of infecting certain types of quiescent cells, there has been significant interest in the application of lentivirus-derived vectors to the transduction of hematopoietic cells. We used the SIV lentiviral system for efficient platelet-targeting gene transduction, because it is potentially safe. The SIV lentiviral system was derived from SIVagmTYO1 and is nonpathogenic to its natural host and to experimentally infected Asian macaques. Replication-competent virus particles were not detected in vector-infected cells, and the risk of development of replication-competent lentivirus particles in HIV carrier patients may be significantly lower than that for the HIV-based vectors. Accordingly, SIV vectors have an advantage in respect to safety issues and in clinical applications of hematopoietic stem cell-directed gene therapy. Furthermore, we used the GPIb promoter for efficient transgene expression in platelets because the promoter activty of GPIb was more potent than that of GPIIb and GPVI in megakaryocytes, and the GPIb promoter works at a later stage of megakaryopoiesis. Platelet-targeting gene therapy using the GPIb promoter was expected to allow more specific and restricted expression in platelets.

Hemophilia A is an X chromosome-linked bleeding disorder caused by defects in the FVIII gene. Hemophilia is considered suitable for gene therapy, because it is caused by a single gene abnormality and therapeutic coagulation factor levels may well vary over in a broad range (5–100%). Although sustained therapeutic expression of FVIII has been achieved in preclinical studies using a wide range of gene transfer technologies targeted at different tissues, the emergence of neutralizing antibodies often limits their clinical applications. The transduction of hematopoietic stem cells is not an exception. Although lentiviral FVIII gene transduction of hematopoietic stem cells is able to produce therapeutic levels of FVIII, the emergence of neutralizing antibodies to FVIII has resulted in decreased levels of FVIII activity. Platelet-directed gene therapy for hemophilia A has a possible advantage for therapeutic applications, because the use of the platelet-specific system may limit the development of inhibitors by preventing the expression of FVIII in antigen presenting cells. Furthermore, 10–30% of populations with hemophilia A develop inhibitors to the infusion products, which leads to the disruption of the coagulation factor and severe bleeding. Under these conditions, platelet-directed gene therapy of hemophilia A is very attractive because platelets could specifically store the protein in the bloodstream and then specifically release it at sites of thrombus formation, thereby minimizing the influence of circulating inhibitors.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5161fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5161fjev1 20/9/1522    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ohmori, T. Articles by Sakata, Y. Search for Related Content PubMed PubMed Citation Articles by Ohmori, T. Articles by Sakata, Y.