SUSTAINED GENE-EXPRESSION IN RETROVIRALLY TRANSDUCED, ENGRAFTING HUMAN HEMATOPOIETIC STEM-CELLS AND THEIR LYMPHO-MYELOID PROGENY

Inefficient retroviral-mediated gene transfer to human hematopoietic s tem cells (HSC) and insufficient gene expression in progeny cells deri ved from transduced HSC are two major problems associated with HSC-bas ed gene therapy. In this study we evaluated the ability of a murine st em cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintai n gene expression in transduced human hematopoietic cells. CD34(+) cel ls lacking lineage differentiation markers (CD34(+)Lin(-)) isolated fr om human bone marrow and mobilized peripheral blood were transduced us ing 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 expres sed a high level of NGFR (NGFR(+)) as assessed by fluorescence-activat ed cell sorter analysis. When these CD34(+)Lin(-)NGFR(+) cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-t erm stromal cultures, sustained reporter expression was observed in di fferentiated erythroid and myeloid cells derived from transduced proge nitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34(+)Lin(-)NGFR(+) cells were used to repopul ate human bone grafts implanted in severe combined immunodeficient mic e, MSCV-directed NGFR expression could be detected on 37% +/- 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. Thes e findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC. (C ) 1998 by The American Society of Hematology.