Repopulation of B-lymphocytes with restricted gene expression using haematopoietic stem cells engineered with lentiviral vectors.
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 (Emu) flanked by the associated matrix attachment regions (MARs) was inserted upstream of the CD19 promoter. Incorporation of the Emu-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.
Taher
T E
TE
Bone 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.
Tulone
C
C
Fatah
R
R
D'Acquisto
F
F
Gould
D J
DJ
Mageed
R A
RA
D000818:Animals; D018941:Antigens, CD19 / Q000235:genetics* / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D005434:Flow Cytometry; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D011401:Promoter Regions, Genetic; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes
D018941:Antigens, CD19; D049452:Green Fluorescent Proteins