Sustained gene expression in retrovirally transduced, engrafting human hematopoietic stem cells and their lympho-myeloid progeny

Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34⁺ cells lacking lineage differentiation markers (CD34⁺Lin^-) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34⁺ cell population retained the Lin^- phenotype after 4 days in culture and at least 30% of these expressed e high level of NGFR (NGFR⁺) as assessed by fluorescence-activated cell sorter analysis. When these CD34⁺Lin-NGFR⁺ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34⁺Lin^-NGFR⁺ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% ± 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.