1. ¹INSERM E 0217, Laboratoire de Pathologie Moléculaire et Thérapie Génique, Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux, France
2. ²UMR CNRS 1456, Laboratoire de Greffe de Moelle, Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux, France
3. ³Service des Maladies du Sang, Hôpital Haut-Lévèque, Pessac, France
4. ⁴Service des Maladies infectieuses Hôpital St Andíe, Bordeaux, France

Correspondence: François Moreau-Gaudry, Fax: (33) 5 56 98 33 48. E-mail: Francois.Moreau-Gaudry@pmtg.u-bordeaux2.fr

Received 26 November 2002; Accepted 7 January 2003.

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell (HSC) disorder in which an acquired somatic mutation of the X-linked PIGA gene results in a deficiency in GPI-anchored surface proteins. Clinically, PNH is dominated by a chronic hemolytic anemia, often associated with recurrent nocturnal exacerbations, neutropenia, thrombocytopenia, and thrombotic tendency. Allogenic bone marrow transplantation is the only potentially curative treatment for severe forms of PNH but is associated with a high treatment-related morbidity and mortality. HSC gene therapy could provide a new therapeutic option, especially when an HLA-matched donor is not available. To develop an efficient gene transfer approach, we have designed a new SIN lentiviral vector (TEPW) that contains the PIGA cDNA driven by the human elongation factor 1 promoter, the central DNA flap of HIV-1, and the WPRE cassette. TEPW transduction led to a complete surface expression of the GPI anchor and CD59 in PIGA-deficient cell lines without any selection procedure. Moreover, efficient gene transfer was achieved in bone marrow and mobilized peripheral blood CD34⁺ cells derived from two patients with severe PNH disease. This expression was stable during erythroid, myeloid, and megakaryocytic liquid culture differentiation. CD59 surface cell expression was fully restored during 5 weeks of long-term culture.