1. ¹Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London, Queen Mary's School of Medicine and Dentistry, University of London, London, UK
2. ²Department of Immunology and Molecular Pathology, University College London, London, UK
3. ³Biochemical Pharmacology, William Harvey Research Institute, Barts and The London, Queen Mary's School of Medicine and Dentistry, University of London, London, UK

Correspondence: Professor RA Mageed, Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London, Queen Mary School of Medicine and Dentistry, University of London, Charterhouse Square, London EC1M 6BQ, UK. E-mail: r.a.mageed@qmul.ac.uk

Received 14 October 2007; Revised 21 December 2007; Accepted 23 January 2008; Published online 20 March 2008.

Abstract

B-lymphocytes play a key role in the pathogenesis of many immune-mediated diseases, such as autoimmune and atopic diseases. Therefore, targeting B-lymphocytes provides a rationale for refining strategies to treat such diseases for long-term clinical benefits and minimal side effects. In this study we describe a protocol for repopulating irradiated mice with B-lymphocytes engineered for restricted expression of transgenes using haematopoietic stem cells. A self-inactivating lentiviral vector, which encodes enhanced green fluorescence protein (EGFP) from the spleen focus-forming virus (SFFV) promoter, was used to generate new vectors that permit restricted EGFP expression in B-lymphocytes. To achieve this, the SFFV promoter was replaced with the B-lymphocyte-restricted CD19 promoter. Further, an immunoglobulin heavy chain enhancer (E) flanked by the associated matrix attachment regions (MARs) was inserted upstream of the CD19 promoter. Incorporation of the E-MAR elements upstream of the CD19 promoter resulted in enhanced, stable and selective transgene expression in human and murine B-cell lines. In addition, this modification permitted enhanced selective EGFP expression in B-lymphocytes in vivo in irradiated mice repopulated with transduced bone marrow haematopoietic stem cells (BMHSCs). The study provides evidence for the feasibility of targeting B-lymphocytes for therapeutic restoration of normal B-lymphocyte functions in patients with B-cell-related diseases.