1. ¹Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA
2. ²Holland Laboratory, Hematopoiesis Department, American Red Cross, Rockville, Maryland 20855, USA
3. ³Department of Hematology and Bone Marrow Transplantation, Charite Hospital, Berlin, Germany
4. ⁴Department of Clinical Genetics and Human Genetics, VU University Medical Center, Amsterdam, Netherlands
5. ⁵Flow Cytometry Facility, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

Correspondence: Christopher E. Walsh, Department of Medicine, Mount Sinai School of Medicine, Box 1079, One Gustave L. Levy Place, New York, NY 10029, USA. Fax: (212) 426-4390. E-mail: Christopher-E.Walsh@msnyuhealth.org

Received 6 April 2003; Accepted 22 June 2003.

Abstract

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure due to defective stem cell function. FA patients' cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC), exposure to which results in cytogenetic aberrations and cell death. To date Moloney murine leukemia virus vectors have been used in clinical gene therapy. Recently, third-generation lentiviral vectors based on the HIV-1 genome have been developed for efficient gene transfer to hematopoietic stem cells. We generated a self-inactivating lentiviral vector expressing the FA group A cDNA driven by the murine stem cell virus U3 LTR promoter and used the vector to transduce side-population (SP) cells isolated from bone marrow of Fanconi anemia group A (Fanca) knockout mice. One thousand transduced SP cells reconstituted the bone marrow of sublethally irradiated Fanca recipient mice. Phenotype correction was demonstrated by stable hematopoiesis following MMC challenge. Using real-time PCR, one proviral vector DNA copy per cell was detected in all lineage-committed cells in the peripheral blood of both primary and secondary recipients. Our results suggest that the lentiviral vector transduces stem cells capable of self-renewal and long-term hematopoiesis in vivo and is potentially useful for clinical gene therapy of FA hematopoietic cells.