TY - JOUR
T1 - Design of Ad5F35 vectors for coordinated dual gene expression in candidate human hematopoietic stem cells.
AU - Na, Manli
AU - Fan, Xiaolong
PY - 2010
Y1 - 2010
N2 - OBJECTIVE: Adenoviral vector mediated gene expression is an attractive approach to manipulate or report gene expression in human hematopoietic stem cells (HSC), when transient gene expression is preferred. Previous studies have demonstrated that fiber retargeted Ad5F35 vectors can mediate efficient gene transfer into human HSCs. In this study, we have investigated the potential of bi-directional promoter controlled Ad5F35 vector for coordinated dual gene expression in candidate HSCs. MATERIAL AND METHODS: We have engineered Ad5F35-DeltaLNGFR-BiDp encoding kinase domain deleted low-affinity NGF receptor (DeltaLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter, which is composed of human phosphoglycerate kinase (PGK) promoter and minimal core promoter from human cytomegalovirus (mCMV). The expression pattern of DeltaLNGFR and GFP following Ad5F35-DeltaLNGFR-BiDp gene transfer in various cell types including candidate HSCs was compared to Ad5F35-DeltaLNGFR-IRES vector encoding PGK promoter controlled bicistronic expression cassette for DeltaLNGFR and GFP. RESULTS AND CONCLUSIONS: Using Ad5F35-DeltaLNGFR-BiDp, we demonstrated a coordinated, high-level dual gene expression in leukemic cells and cord blood CD34(+) cells. However, the ability of Ad5F35-DeltaLNGFR-BiDp-GFP for coordinated dual gene expression varied significantly between re-populating progenitor cells. In NOD/SCID mice bone marrow transplantation assay, sorted CD34(+)/DeltaLNGFR(+)/GFP(+) cells following infection with Ad5F35-DeltaLNGFR-BiDp showed predominantly myeloid lineage reconstitution with limited lymphoid lineage differentiation capacity, whereas the CD34(+)/DeltaLNGFR(+)/GFP(-) cells exhibited both myeloid and lymphoid reconstitution. This study indicates that bi-directional promoter controlled Ad5F35 vector such as Ad5F35-DeltaLNGFR-BiDp can be particularly useful for manipulation of myeloid progenitor cells and potentially also in myeloid lineage leukemic cells.
AB - OBJECTIVE: Adenoviral vector mediated gene expression is an attractive approach to manipulate or report gene expression in human hematopoietic stem cells (HSC), when transient gene expression is preferred. Previous studies have demonstrated that fiber retargeted Ad5F35 vectors can mediate efficient gene transfer into human HSCs. In this study, we have investigated the potential of bi-directional promoter controlled Ad5F35 vector for coordinated dual gene expression in candidate HSCs. MATERIAL AND METHODS: We have engineered Ad5F35-DeltaLNGFR-BiDp encoding kinase domain deleted low-affinity NGF receptor (DeltaLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter, which is composed of human phosphoglycerate kinase (PGK) promoter and minimal core promoter from human cytomegalovirus (mCMV). The expression pattern of DeltaLNGFR and GFP following Ad5F35-DeltaLNGFR-BiDp gene transfer in various cell types including candidate HSCs was compared to Ad5F35-DeltaLNGFR-IRES vector encoding PGK promoter controlled bicistronic expression cassette for DeltaLNGFR and GFP. RESULTS AND CONCLUSIONS: Using Ad5F35-DeltaLNGFR-BiDp, we demonstrated a coordinated, high-level dual gene expression in leukemic cells and cord blood CD34(+) cells. However, the ability of Ad5F35-DeltaLNGFR-BiDp-GFP for coordinated dual gene expression varied significantly between re-populating progenitor cells. In NOD/SCID mice bone marrow transplantation assay, sorted CD34(+)/DeltaLNGFR(+)/GFP(+) cells following infection with Ad5F35-DeltaLNGFR-BiDp showed predominantly myeloid lineage reconstitution with limited lymphoid lineage differentiation capacity, whereas the CD34(+)/DeltaLNGFR(+)/GFP(-) cells exhibited both myeloid and lymphoid reconstitution. This study indicates that bi-directional promoter controlled Ad5F35 vector such as Ad5F35-DeltaLNGFR-BiDp can be particularly useful for manipulation of myeloid progenitor cells and potentially also in myeloid lineage leukemic cells.
U2 - 10.1016/j.exphem.2010.03.007
DO - 10.1016/j.exphem.2010.03.007
M3 - Article
SN - 1873-2399
VL - Apr 8
SP - 446
EP - 452
JO - Experimental Hematology
JF - Experimental Hematology
ER -