Long-Term Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice with Human CD34 + Cells Transduced by a Self-Inactivating Human Immunodeficiency Virus Type 1 Vector

Abstract

Human hematopoietic cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous inherited or acquired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with human CD34⁺ cells transduced by an HIV-1-based self-inactivating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34⁺ cells were transduced (up to 76%) at a low multiplicity of infection (MOI of 5) in the absence of cytokine prestimulation. Introduction of transduced hCD34⁺ cells into irradiated recipients resulted in multilineage engraftment and stable transgene expression for 18 weeks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45⁺ cells and up to 63% hCD45⁺/EGFP⁺ cells. Analysis of extramedullar 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⁺ colony-forming cells(CFCs) by fluorescence microscopy and by PCR analysis of provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hematopoietic stem cell gene transfer and provide strong support for their future clinical evaluation.