title: Sustained gene expression in retrovirally transduced, engrafting human hematopoietic stem cells and their lympho-myeloid progeny.
abstract: 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 a 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.
authors.affiliation: SyStemix, Inc, Palo Alto, CA, USA. LCheng@Osiristx.com
authors.forename: L
authors.initials: L
authors.lastname: Cheng
authors.forename: C
authors.initials: C
authors.lastname: Du
authors.forename: C
authors.initials: C
authors.lastname: Lavau
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Tong
authors.forename: B P
authors.initials: BP
authors.lastname: Chen
authors.forename: R
authors.initials: R
authors.lastname: Scollay
authors.forename: R G
authors.initials: RG
authors.lastname: Hawley
authors.forename: B
authors.initials: B
authors.lastname: Hill
mesh_terms: D000818:Animals; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006085:Graft Survival; D006410:Hematopoiesis / Q000502:physiology; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology*; D006801:Humans; D007962:Leukocytes / Q000166:cytology / Q000502:physiology; D051379:Mice; D016513:Mice, SCID; D017475:Receptors, Nerve Growth Factor / Q000235:genetics; D012190:Retroviridae
chemical_list: D017475:Receptors, Nerve Growth Factor