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.

High Levels of Transgene Expression Following Transduction of Long‐Term NOD/SCID‐Repopulating Human Cells with a Modified Lentiviral Vector

“…Human 293T and TE671 cells were used to produce viruses after transfection, while the three adherent cell lines were target cells to monitor viral transduction. Human CD34 + (suspension) TF1 cells (ATCC CRL-2003) were maintained in RPMI 1640 plus 10% FBS and 1 ng/ml GM-CSF (Amgen), and used as previously described to monitor hematopoietic cell transduction [3].…”

“…One such LTR is from the murine stem cell virus (MSCV), a genetically optimized RV based on MESV and Mo-MLV [2]. Using the MSCV-based MGIN RV packaged with either amphotropic or GALV envelopes, we reported high levels of transgene expression in the progeny of transduced human SRC in the bone marrow (BM) of transplanted mice [1,[3][4]. Similar results were reported by other investigators using RV similar to MGIN and the same NOD/SCID mouse model [5][6][7], and in a nonhuman primate transplantation model [8].…”

“…This may explain why transgene expression level was low in human hematopoietic progenies derived from SRC transduced with an LV vector containing the CMV IE promoter [10]. Since the MSCV LTR has been shown to function as an effective promoter in SRC [1,[3][4][5][6][7], we decided to evaluate a hybrid LV in which the HIV-1 LTR was replaced in part with sequences from the MSCV LTR.…”

“Rainbow” Reporters for Multispectral Marking and Lineage Analysis of Hematopoietic Stem Cells

“…Whereas vital staining procedures maintain cell function, there is a requirement to transport sufficient concentrations of fluorogenic substrates across cell membranes and retain the cleaved fluorescent products intracellularly. Genes encoding cell-surface proteins, including murine CD24 and the low-affinity human nerve growth factor receptor, have also been utilized to enumerate gene transfer efficiency and mark HSCs [14,15]. But a potential disadvantage of this approach includes the possibility of changing the biological properties of the transduced HSCs due to aberrant triggering of signal transduction pathways or alteration of their homing properties.…”

Directed differentiation of embryonic stem cells: Genetic and epigenetic methods

“…commitment. 136 Exposure to Wnt3 protein was shown to increase in the number of EBs containing hematopoietic One concern with these approaches is that it is unclear what the remaining cell types are, because authors foci, 54 suggesting new signaling pathways which may be involved in early lineage segregation of hematopoietic present RT-PCR data to show expression of targeted genes, but not of stem cell markers (to address culture progenitors. Other mesodermal derivatives, particularly muscle cells, are described in the forced differentiation purity) or markers of other phenotypes.…”