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TECHNOLOGY DEVELOPMENT

Glycoprotein Ib Promoter Drives Megakaryocytic Lineage-Restricted Expression After Hematopoietic Stem Cell Transduction Using a Self-Inactivating Lentiviral Vector

^aInstitut Cochin, Département d'Hématologie, Paris, France;
^bInstitut Cochin, Plateforme de Microscopie Électronique, Paris, France;
^cInstitut National de la Santé et de la Recherche Médicale, U567, Paris, France;
^dCentre National de la Recherche Scientifique, Paris, France;
^eUniversité Paris Descartes, Paris, France;
^fService d'Hématologie Biologique, Hôpital Henri Mondor Université Paris XII, Créteil, France

Key Words. Megakaryocytes • Lentiviral vectors • Promoter • Human CD34+ cells • Gene transfer • Gene expression • Platelet Glycoprotein Ib

Correspondence: Anne Dubart-Kupperschmitt, M.D., Ph.D., Institut Cochin, Department of Hematology, Hôpital de Port-Royal, 123 Bd de Port-Royal, Paris 75014, France. Telephone: 33-153104369; Fax: 33-143251167; e-mail: dubart{at}cochin.inserm.fr

Received May 26, 2006; accepted for publication March 12, 2007.
First published online in STEM CELLS EXPRESS   March 22, 2007.



Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short-lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34⁺ hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral-blood mobilized CD34⁺ cells were transduced with a human immunodeficiency virus-derived self-inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIb, GpIIb, or EF1 gene regulatory sequences. Both GpIb and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIb promoter was strictly MK-specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328-base-pair fragment of the human GpIb gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34⁺ hematopoietic cells.

Disclosure of potential conflicts of interest is found at the end of this article.

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