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Integrin Iib Promoter-Targeted Expression of Gene Products In Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9654–9659, August 1999 Cell Biology Integrin ␣IIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells ʈ DAVID A. WILCOX*†,JOHN C. OLSEN*‡,LORI ISHIZAWA§,MICHAEL GRIFFITH§, AND GILBERT C. WHITE II*†¶ ¶Center for Thrombosis and Hemostasis, Departments of *Medicine and †Pharmacology, and ‡Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599; and §Nexell Therapeutics Inc., Irvine, CA 92618 Communicated by Inder M. Verma, The Salk Institute for Biological Studies, San Diego, CA, June 17, 1999 (received for review March 26, 1999) ABSTRACT Megakaryocyte-specific expression of the blood coagulation, and release granule contents. This regu- platelet-adhesion receptor, integrin ␣IIb␤3, is caused by the lates blood clotting, stimulates wound healing, and mediates presence of regulatory elements of the ␣IIb promoter that platelet aggregation to seal damaged vessels. direct high-level, selective gene transcription early in Megakaryocyte-specific expression of the major platelet- megakaryocytopoiesis. To develop methods for targeted ex- aggregation receptor, integrin ␣IIb␤3, is caused by the pres- pression of transgenes, we transduced human CD34؉ periph- ence of regulatory elements of the ␣IIb promoter that direct eral blood cells with a murine leukemia virus (MuLV) vector high-level, selective gene transcription early in megakaryocy- controlled by the human integrin ␣IIb promoter (nucleotides topoiesis. The ␣IIb promoter has been previously demon- -؊889 to ؉35). A naturally occurring cDNA encoding the PlA2 strated to direct high-level, megakaryocyte-targeted gene tran alloantigen form (Pro33) of the integrin ␤3 subunit was scription in human cell lines (1–3), rat cells (4), and transgenic subcloned into this construct (؊889PlA2␤3) and transduced mice (5, 6). An 800-nt fragment of the human ␣IIb promoter A1 into cells that endogenously synthesized Pl ␤3 (Leu33)asa directed expression of the thymidine kinase gene in a marker for detection of provirus-derived ␤3. The ability of this megakaryocyte-selective manner in the transgenic mice stud- vector to target expression of PlA2␤3 to megakaryocytes was ies (5, 6). This promoter fragment binds GATA and Ets factors first examined in cell lines. Immunoblot analysis with human to induce a high level of gene transcription (7), which is anti-PlA2 alloserum detected synthesis of PlA2␤3 in trans- restricted to developing megakaryocytes because of an ele- duced promegakaryocytic cells; however, PlA2␤3 protein was ment localized to the immediate 5Ј upstream region of the ␣IIb not detected in transduced epithelial cells. Human hemato- gene between nucleotides Ϫ80 and Ϫ130 (2–4). -(poietic CD34؉ cells were transduced with ؊889PlA2␤3 virions This investigation uses murine leukemia virus (MuLV and induced to differentiate with megakaryocyte growth and derived vectors driven by an 889-nt fragment of the promoter development factor. A hybrid ␣IIb␤3 complex was formed in of the human ␣IIb gene to induce early and specific transgene progeny megakaryocytes where provirus-derived PlA2␤3 was expression during megakaryocytopoiesis of human cells. Hu- detected associated with endogenous ␣IIb subunit. Another man CD34ϩ hematopoietic cells transduced with MuLV- (IIb promoter-driven MuLV vector (؊889nlacZ) encoding derived vectors encoding either ␤3or␤-galactosidase (␤-gal␣ Escherichia coli ␤-galactosidase was used to demonstrate that demonstrated lineage-specific transgene expression after dif- transgene expression was selectively targeted to the ferentiation along a megakaryocytic pathway (8). The platelet megakaryocyte progeny of transduced CD34؉ cells. These alloantigen 2 (PlA2) form of the integrin ␤3 subunit was used studies demonstrate the feasibility of using ␣IIb promoter- to distinguish provirus-derived protein from endogenous pro- driven MuLV vectors for gene transfer of hematopoietic tein in cultured megakaryocytes. This result demonstrates the CD34؉ cells to target transgene expression in developing feasibility of lineage-specific gene expression in pluripotent megakaryocytes and platelets and indicate potential applica- hematopoietic stem cells and has implications for human gene tions toward human gene therapy for platelet disorders. therapy of hematopoietic disorders. During megakaryocytopoiesis, pluripotent hematopoietic pro- MATERIALS AND METHODS genitor and precursor cells differentiate to mature polyploid megakaryocytes that shed small anucleate platelets. This pro- Antibodies. Monoclonal antibody AP3 (9) and polyclonal ␤ cess is associated with three remarkable cellular events. First, antibodies specific for 3 were from Peter J. Newman (Blood A2 ␤ megakaryocyte-specific adhesion receptors are expressed, Research Institute, Milwaukee, WI). Human anti-Pl 3 namely integrin ␣IIb␤3 and the glycoprotein Ib-V–IX com- alloserum was from Brian Curtis (Blood Center of Southeast- plex, which mediate platelet–platelet and platelet–extracellu- ern Wisconsin, Milwaukee, WI). Monoclonal antibody AP2 ␣ ␤ lar matrix interactions. Second, cytoplasmic granules are (10), which recognizes the IIb 3 complex was from Robert R. formed that contain agonists, hemostatic mediators, and Montgomery (Blood Research Institute, Milwaukee, WI). A growth factors. Third, signaling pathways develop that produce monoclonal antibody that recognizes the erythrocyte-specific cyclic nucleotides, inositol phosphates, and endoperoxides, protein, glycophorin A (GPA), was from Sigma. which induce release of granule components and activation of Cell Lines. Dami and human 293 cell lines were from the membrane receptors. Interactions between membrane recep- American Type Culture Collection. CFT1 cells were previously described (11). tors, cytoplasmic granules, and signaling pathways induce ␣ platelets to bind to adhesive proteins exposed on damaged IIb Promoter. Genomic DNA was isolated from the human blood vessels, complex with plasma components, mediate promegakaryocyte cell line, Dami, and a fragment of the ␤ ␤ The publication costs of this article were defrayed in part by page charge Abbreviations: -gal, -galactosidase; GPA, glycophorin A; rhIL, recombinant human IL; MuLV, murine leukemia virus; LTR, long payment. This article must therefore be hereby marked ‘‘advertisement’’ in terminal repeat; CMV, cytomegalovirus. accordance with 18 U.S.C. §1734 solely to indicate this fact. ʈTo whom reprint requests should be addressed. E-mail: gcwhite@ PNAS is available online at www.pnas.org. med.unc.edu. 9654 Downloaded by guest on September 30, 2021 Cell Biology: Wilcox et al. Proc. Natl. Acad. Sci. USA 96 (1999) 9655 human ␣IIb gene promoter was amplified by PCR using sense The sequence of pϪ889nLacZ was confirmed by nucleotide primer ‘‘Ϫ889’’ (5Ј-TTACGCGTCGACAGATCTGTGCT- analysis. CAATGCTGTGCC-3Ј) from nucleotide Ϫ889 to Ϫ872 (bold- pCMVnLacZ. pCMVnLacZ was constructed when a cassette face) of ␣IIb and antisense primer (5Ј-ATAGTTTAGCGGC- encoding the cytomegalovirus (CMV) immediate early pro- CGCGCTCGCATCTTCCTTCTTCCAC-3Ј) encoding nucle- moter and enhancer region and E. coli ␤-gal was removed from otides ϩ32 to ϩ22 of ␤3 and nucleotides ϩ35 to ϩ19 of ␣IIb. pCMVnlac with restriction enzymes (NaeI and SamI) and The antisense primer positions the ␣IIb promoter in-frame inserted between the LTRs of pHIT-SIN. with the translation start site of ␤3. An ␣IIb promoter used for Retroviral Helper Plasmids. Plasmids pCI-GPZ and pCI- ␤-gal gene transcription was constructed with the Ϫ889 primer VSV-G were previously described (16). A and antisense primer (5Ј-ATTATTGGGCCCCATCTTCCT- Typing of Pl Alloantigen. All cell samples used for Ϫ A2␤ TCTTCCAC-3Ј) encoding nucleotides ϩ4048 to ϩ4058 of 889Pl 3 transduction were confirmed homozygous for the A1␤ ␤-gal in pCMVnlac (12) and nucleotides ϩ35 to ϩ19 of ␣IIb. Pl 3 alloantigen as described (17). Retrovirus Production. Human 293 cells were transiently PCR products were cloned into plasmid vectors pGL3 or ␮ pGEM-7zf (ϩ) (Promega), and sequence was confirmed by transfected on 10-cm plates with 15 g each of pCI-GPZ, Ϫ A2␤ Ϫ nucleotide analysis (13). pCI-VSV-G, and either p 889Pl 3, p 889nLacZ, or pC- Retroviral Constructs. p-889PlA2␤3. The Ϫ889 ␣IIb pro- MVnLacZ by using the calcium transfection system (Life moter for ␤3 expression was cloned 5Ј of cDNA encoding the Technologies, Gaithersburg, MD). After 12 h, cells were n platelet alloantigen 2 (PlA2) form of ␤3 (14) (from Peter J. placed in fresh medium containing 10 mM -butyric acid (Sigma) (18), and incubation continued at 37°C for 24 h. Newman) within Bluescript plasmid vector (Stratagene). Virions were concentrated 500-fold, resuspended in Iscove’s Briefly, PlA2␤3 cDNA in Bluescript was treated with restriction modified Dulbecco’s Eagle’s medium (IMDM), and stored at enzymes (NotI, NsiI, and SalI), and a three-way ligation was Ϫ80°C. Replication-competent virions were confirmed absent performed to join the NotI and SalI ␣IIb promoter fragment A2␤ Ϫ A2␤ from viral preparations by using extended marker rescue assays and the two Pl 3-Bluescript fragments. The 889Pl 3 as described (18). DNA cassette (Fig. 1) was removed from Bluescript by treat- Selection of CD34؉ Cells. Peripheral blood cells were ment with XbaI and ligated between the long terminal repeats collected after obtaining written informed consent from nor- (LTR) of the MuLV-derived retroviral vector, pHIT-SIN, to mal healthy subjects
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