Long-term engraftment of nonobese diabetic/severe combined immunodeficientmice with human CD34(+) cells transduced by a self-inactivating human immunodeficiency virus type 1 vector

Human hematopoietic cells with in vivo repopulating potential hold much pro mise as a target for corrective gene transfer for numerous inherited or acq uired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SC ID) mice with human CD34(+) cells transduced by an HIV-1-based self-inactiv ating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34(+) cells were transduced (up to 76%) at a low mul tiplicity of infection (MOI of 5) in the absence of cytokine prestimulation . Introduction of transduced hCD34(+) cells into irradiated recipients resu lted in multilineage engraftment and stable transgene expression for 18 wee ks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45(+) cells and up to 63% hCD45(+)/EGFP(+) cells. Analysis of extram edullar splenic reconstitution showed up to 13% hCD45(+) cells and up to 41 % hCD45(+)/EGFP(+) cells. Analysis of human progenitor cells isolated from bone marrow of recipient animals showed equivalent percentages of EGFP(+) c olony-forming cells (CFCs) by fluorescence microscopy and by PCR analysis o f provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hemat opoietic stem cell gene transfer and provide strong support for their futur e clinical evaluation.